Recombinant modified vaccinia virus ankara (MVA) respiratory syncytial virus (RSV) vaccine

ABSTRACT

Provided herein are recombinant modified vaccinia virus Ankara (MVA) strains as improved vaccines against infection with Respiratory Syncytial Virus (RSV virus) and to related products, methods and uses. Specifically, provided herein are genetically engineered recombinant MVA vectors comprising at least one nucleotide sequence encoding an antigenic determinant of an RSV membrane glycoprotein and at least one nucleotide sequence encoding an antigenic determinant of an RSV nucleocapsid protein. Also provided herein are products, methods and uses thereof, e.g., suitable to affect an immune response in a subject, or suitable to diagnose an RSV infection, as well as to determine whether a subject is at risk of recurrent RSV infection.

This application is a National Phase application under 35 U.S.C. §371 ofInternational Application No. PCT/EP2013/055483, filed Mar. 15, 2013,and claims the benefit under 35 U.S.C. §365 of European Application1200594.2 filed Aug. 1, 2012, and the benefit under 35 U.S.C. §119(e) ofU.S. Provisional Patent Application 61/678,367 filed Aug. 1, 2012, thedisclosures of which are incorporated by reference herein in theirentirety.

FIELD

The present invention relates to a recombinant modified vaccinia virusAnkara (MVA virus) as an improved vaccine against an infection withRespiratory Syncytial Virus (RSV virus) and to related products, methodsand uses. Specifically, the present invention relates to a geneticallyengineered recombinant MVA vector comprising at least one nucleotidesequence encoding an antigenic determinant of an RSV membraneglycoprotein and at least one nucleotide sequence encoding an antigenicdeterminant of an RSV nucleocapsid protein. The invention also relatesto products, methods and uses thereof, e.g., suitable to affect animmune response in a subject, or suitable to diagnose an RSV infection,as well as to determine whether a subject is at risk of recurrent RSVinfection.

BACKGROUND

RSV is a significant respiratory pathogen. Acute lower respiratory tract(LRT) infection causes significant morbidity and mortality in infantsand children under the age of five years worldwide [A. M. Aliyu et al.(2010), Bayero J. Pure Appl. Sci. 3(1):147-155]. Respiratory syncytialvirus (RSV) is the most clinically important cause of LRT infection;primary infection with RSV generally occurs by age 2 [W. P. Glezen(1987), Ped. Virol. 2:1-4; Y. Murata (2009), Clin. Lab. Med.29(4):725-739]. Because primary RSV infection does not induce completeimmunity to RSV, frequent re-infections occur throughout life, with themost severe infections developing in the very young, the very old, andin immune-compromised patients of any age [Y. Murata (2009)].

As many of 40% of those infected with RSV eventually develop serious LRTdisease requiring hospitalization, with the severity and intensity ofthe disease depending on the magnitude and intensity of infection andthe host response [Aliyu et al. (2010)]. RSV can also cause serious LRTdisease in patients of any age having compromised immune, respiratory,or cardiac systems, and may also predispose children to laterdevelopment of asthma. In the United States alone, RSV causes anestimated 126,000 hospitalizations and 300 infant deaths a year [Y.Murata (2009)]. Furthermore, RSV accounts for more than 80,000hospitalizations and more than 13,000 deaths each winter among elderlypatients, and those with underlying cardiopulmonary or immunosuppressiveconditions [Y. Murata (2009)]. Despite the importance of RSV as arespiratory pathogen, however, there is currently no safe and effectiveRSV vaccine on the market.

RSV is an enveloped RNA virus of the family Paramyxoviridae, subfamilyPneumovirinae [Aliyu et al. (2010)]. Each RSV virion contains anon-segmented, negative-sense, single-stranded RNA molecule ofapproximately 15,191 nucleotides containing ten genes encoding elevenseparate proteins (M2 contains two open reading frames), including eightstructural (G, F, SH, M1, N, P, M2.1, and L) and three non-structuralproteins (NS1, NS2, and M2.2) [Y. Murata (2009)]. The genome istranscribed sequentially from NS1 to L, in the following order:3′-NS1-NS2-N-P-M1-SH-G-F-M2.1-M2.2-L-5′.

The viral envelope contains three transmembrane glycoproteins(attachment glycoprotein (G), fusion glycoprotein (F), and smallhydrophobic protein (SH)), as well as the matrix (M1) protein [Y. Murata(2009)]. During RSV replication, the virus first attaches to the targetcell in a process mediated by the heavily glycosylated G protein. Thevirus then fuses with the host cell in a process mediated by the Fprotein, thereby penetrating the cell membrane and entering the hostcell; the F protein is also required for the formation of the syncytiacharacteristic of RSV-infected cells. The attachment and fusionprocesses are augmented by SH protein. The M1 protein regulates theassembly of mature RSV by interacting with the envelope proteins F and Gand with the nucleocapsid proteins N, P, and M2.1 (see below). Withinthe envelope, viral RNA is encapsidated by a transcriptase complexconsisting of the nucleocapsid protein (N), phosphoprotein (P),transcription elongation factor (M2.1) and RNA polymerase (L) proteins[Y. Murata (2009)]. N associates with the genomic RNA, while P is acofactor for L, the viral RNA polymerase. M2.1 is an elongation factornecessary for viral transcription, and M2.2 regulates transcription ofthe viral genome. Finally, NS1 and NS2 inhibit type I interferonactivity.

Clinical RSV isolates are classified according to antigenic group (A orB) and further subdivided into multiple genotypes (e.g., A2 or A_(Long)for the A group; and B1, CH-18537, or 8/60 for the B group) based on thegenetic variability within the viral genome of each antigenic group [Y.Murata (2009)]. Classification is based on the reactivity of the viruseswith monoclonal antibodies directed against the attachment glycoprotein(G protein) and by various genetic analyses. [M. Sato et al., J. Clin.Microbiol. 43(1):36-40 (2005)]. Among viral isolates, some RSV-encodedproteins are highly conserved at the level of amino acid sequence (e.g.,F), while others vary extensively (e.g., G) between and within the twomajor antigenic groups [Y. Murata (2009)]. The F proteins from the A andB antigenic groups share considerable homology. In contrast, the Gprotein differs considerably between the two antigenic groups.

The G protein is the most variable RSV protein, with its hypervariableC-terminal region accounting for most of the strain-specific epitopes.The molecular epidemiology and evolutionary patterns of G protein haveprovided important information about the clinical and epidemiologicalfeatures of RSV. Typically several different genotypes circulate atonce, and the one that predominates in a community every year maychange. However, the importance of strain diversity to the clinical andepidemiological features of RSV remains poorly understood. RecombinantRSV proteins are therefore accompanied by a strain designation toindicate the original RSV strain from which the gene or protein wascloned. For example, a cloned G protein from RSV strain A_(Long) isdesignated G(A_(Long)), RSV A_(Long) G, or RSV A_(Long) G protein.

RSV stimulates a variety of immune responses in infected hosts,including the secretion of chemokines and cytokines, production ofneutralizing humoral and mucosal antibodies, and production of CD4+(e.g., T_(H)1 and T_(H)2) and CD8+ (e.g., CTL) T-cells. Such host immuneresponses are largely responsible for the clinical manifestations of RSVinfection, since the virus causes limited cell cytopathology in vivo [Y.Murata (2009)]. The phenotypic manifestations and severity ofRSV-induced disease are apparently mediated by the balance andinteractions among the range of immune responses stimulated by RSVinfection [Y. Murata (2009)].

Many previous studies suggest that the cellular and humoral immuneresponses play different roles in the induction of immunity to RSV andthe resolution of RSV infection, as well as in disease progression [Y.Murata (2009) and references therein]. For example, studies with ahumanized anti-F antibody showed that while anti-RSV antibodies aresufficient to prevent or limit the severity of infection, they are notrequired for clearing viral infection [Y. Murata (2009); A. F. G.Antonis et al. (2007), Vaccine 25:4818-4827]. In contrast, T-cellresponses are necessary for clearing established RSV infections [Y.Murata (2009)]. The RSV-induced T-cell response also plays a key role inpulmonary pathology during infection. For example, interferon-γ(IFNγ)-secreting T_(H)1 cells—with or without an associated CD8+ CTLresponse—clear RSV with minimal lung pathology, while interleukin 4(IL-4)-secreting T_(H)2 cells also clear RSV, but frequently accompaniedby significant pulmonary changes, including eosinophilic infiltration, ahallmark of the enhanced disease observed during previous vaccine trials(see below).

Despite the abundance of information available regarding the immunology,virology, and physiology of RSV infection, however, it remains far fromclear precisely what sort of immune response is likely to be mosteffective at inducing lasting immunity while also not producing enhanceddisease on post-vaccination exposure to RSV, as discussed in more detailin the following sections.

Prior Vaccine Development

Vaccines typically use one of several strategies to induce protectiveimmunity against a target infectious agent or pathogen (e.g., a virus,bacterium, or parasite), including: (1) inactivated pathogenpreparations; (2) live attenuated pathogen preparations, includinggenetically attenuated pathogen strains; (3) purified protein subunitvaccine preparations; (4) viral vector-based vaccines encoding pathogenantigens and/or adjuvants; and (5) DNA-based vaccines encoding pathogenantigens.

Initial RSV vaccine development efforts focused on an inactivated viruspreparation, until a clinical trial testing efficacy of aformalin-inactivated RSV (FI-RSV) vaccine was conducted in the UnitedStates during the 1960s with disastrous results [M. R. Olson & S. M.Varga (2007), J. Immunol. 179:5415-5424]. A significant number ofvaccinated patients developed enhanced pulmonary disease characterizedby eosinophil and neutrophil infiltrations and a substantialinflammatory response after subsequent natural infection with RSV [Olson& Varga (2007), [Blanco J C et al. (2010) Hum Vaccin. 6:482-92]. Many ofthose patients required hospitalization and a few critically illpatients died. Consequently, investigators began searching for viraland/or host factors contributing to the development of enhanced diseaseafter subsequent challenge in an effort to develop a safer RSV vaccine.That search has yielded much new information about RSV biology and thebroad spectrum of immune responses it can induce, but a safe andeffective RSV vaccine remains elusive.

Post-FI-RSV vaccine development efforts have focused in large part onsingle antigen vaccines using G, F, and, to a lesser extent, N or M2,with the viral antigens delivered either by viral or plasmid DNA vectorsexpressing the viral genes or as purified proteins. [See, e.g., W.Olszewska et al. (2004), Vaccine 23:215-221; G. Taylor et al. (1997), J.Gen. Virol. 78:3195-3206; and L. S. Wyatt et al. (2000), Vaccine18:392-397]. Vaccination with a combination of F+G has also been testedin calves, cotton rats and BALB/c mice with varying results [Antonis etal. (2007) (calves); B. Moss, U.S. patent application Ser. No.06/849,299 (‘the '299 application’), filed Apr. 8, 1986 (cotton rats);and L. S. Wyatt et al. (2000) (BALB/c mice)]. Both F and G areimmunogenic in calves, mice, cotton rats, humans, and to at least somedegree in infant macaques [A. F. G. Antonis et al. (2007) (calves); B.Moss, the '299 application (cotton rats); L. de Waal et al. (2004),Vaccine 22:923-926 (infant macaques); L. S. Wyatt et al. (2000) (BALB/cmice); Y. Murata (2009) (humans)].

Significantly, however, the nature and type of immune response inducedby RSV vaccine candidates varies—often quite considerably—depending onthe type of vaccine used, the antigens selected, the route ofadministration, and even the model organism used. For example,immunization with live RSV or with replicating vectors encoding RSV Fprotein induces a dominant T_(H)1 response accompanied by production ofneutralizing anti-F antibodies and CD8+ CTLs, both associated withminimal pulmonary pathology upon post-vaccination virus challenge [Y.Murata (2009) and references cited therein]. In contrast, immunizationwith an FI-inactivated RSV preparation induces a dominant T_(H)2response completely lacking a CD8+ CTL response, which producesincreased pathological changes in the lungs [Y. Murata (2009) andreferences cited therein]. Interestingly, the administration of RSV Gprotein as a purified subunit vaccine or in a replicating vector inducesa dominant T_(H)2 response eventually producing eosinophilic pulmonaryinfiltrates and airway hyper-reactivity following post-vaccination viruschallenge, a response very similar to the enhanced disease observed withFI-RSV [Y. Murata (2009) and references cited therein]. In addition,while vaccination with modified vaccinia virus Ankara (MVA) encoding RSVF protein induced anti-F antibodies and F-specific CD8+ T-cells incalves, vaccination with MVA-F+MVA-G induced anti-F and anti-Gantibodies but no F- or G-specific CD8+ T-cells [A. F. G. Antonis et al.(2007)].

Vaccination of mice with vaccinia virus (VV) expressing F protein (VV-F)induced a strong CD8+ T-cell response which lead to clearance ofreplicating RSV from lung accompanied by a similar or greater weightloss than mice immunized with FI-RSV [W. Olszewska et al. (2004)].However it was not related to the enhanced disease induced by FI-RSV orVV expressing G protein (VV-G) (combined T_(H)2 response lungeosinophilia and weight loss) resulting from enhanced secretion ofT_(H)2 cytokines such as IL-4 and IL-5. Some in the field suggested thatan RSV vaccine capable of inducing a relatively balanced immune responseincluding both a cellular and a humoral component would be less likelyto display enhanced immunopathology on post-vaccination challenge [W.Olszewska et al. (2004)].

However, while vaccination of BALB/c mice with modified vaccinia virusAnkara (MVA) encoding F, G, or F+G induced just such a balanced immuneresponse, including both a humoral response (i.e., a balanced IgG1 andIgG2a response) and a T_(H)1 response (i.e., increased levels ofIFNγ/interleukin-12 (IL-12) and decreased levels of interleukin-4(IL-4)/interleukin-5 (IL-5)), vaccinated animals nevertheless stilldisplayed some weight loss [W. Olszewska et al. (2004)].

Despite expending considerable effort to characterize the nature andextent of the immune responses induced by various vaccine candidates inseveral different model systems, it remains unclear precisely what sortof immune response is required to convey lasting and complete immunityto RSV without predisposing vaccine recipients to enhanced disease.Because of the marked imbalance between the clinical burden of RSV andthe available therapeutic and prophylactic options, development of anRSV vaccine remains an unmet medical need.

DESCRIPTION

While prior unsuccessful efforts to develop an RSV vaccine focusedprimarily on vaccination with either RSV-F or RSV-G membraneglycoprotein or both, the present inventors have discovered thatvaccination with a recombinant vaccinia virus Ankara (MVA) expressing atleast one antigenic determinant of an RSV membrane glycoprotein and atleast one antigenic determinant of an RSV nucleocapsid protein inducesbetter protection. In addition, such constructs induce almost completesterile immunity when applied by the intranasal route compared tosubcutaneous application, or even when compared to the intramuscularroute of administration used by Wyatt and colleagues [L. S. Wyatt et al.(2000)]. Enhanced protection can be obtained by administering candidateRSV vaccines intranasally in comparison to intramuscular administration.

With recombinant MVAs expressing either RSV F or RSV G membraneglycoprotein (or both) (e.g., MVA-mBN199B) or with recombinant MVAsexpressing at least one antigenic determinant of an RSV membraneglycoprotein and at least one antigenic determinant of an RSVnucleocapsid protein (e.g., MVA-mBN201B), the present inventors observedno replicating RSV in the lung 4 days post-challenge, although RSVgenomes were still detectable by RT-qPCR. Recombinant MVAs expressing atleast one antigenic determinant of an RSV membrane glycoprotein and atleast one antigenic determinant an RSV nucleocapsid protein (e.g.,MVA-mBN201B) induced better protection and a larger decrease in the RSVviral load detectable by RT-qPCR because they induced a stronger CD8+ Tcell response against the antigenic determinant of an RSV nucleocapsidprotein. Administration of such recombinant viruses by the intranasalroute furthermore induced almost complete sterile immunity (almost noRSV viral load detectable by RT-qPCR) because they induced the mucosalimmune response and IgA antibody secretion, responses which were absentwhen such constructs were administered subcutaneously.

In contrast to FI-RSV, such constructs induce a balanced Th1-immuneresponse generating good antibody responses, as well as strong, specificcellular immune responses to the RSV antigens. With intranasaladministration of the vaccine producing IgG antibody levels even higherthan those resulting from conventional subcutaneous administration inaddition to the induction of a good IgA antibody response, protection isimproved and body weight loss reduced. The magnitude of the cellularimmune response was independent of the route of administration, however.Interestingly, the inventors observed a pattern of T-cell responseinduced by recombinant MVAs expressing at least one heterologousnucleotide sequence encoding an antigenic determinant of an RSV membraneglycoprotein and at least one heterologous nucleotide sequence encodingan antigenic determinant of an RSV nucleocapsid protein (e.g.,MVA-mBN201B, expressing RSV F, G, N, and M2 proteins) that was similarto the T-cell response induced by RSV administrations, albeit muchhigher.

Thus, in a first aspect, the present invention provides a recombinantmodified vaccinia virus Ankara (MVA) comprising at least one nucleotidesequence encoding an antigenic determinant of a respiratory syncytialvirus (RSV) membrane glycoprotein and at least one nucleotide sequenceencoding an antigenic determinant of an RSV nucleocapsid protein.

Modified Vaccinia Virus Ankara (MVA)

MVA has been generated by more than 570 serial passages on chickenembryo fibroblasts of the dermal vaccinia strain Ankara [Chorioallantoisvaccinia virus Ankara virus, CVA; for review see Mayr et al. (1975),Infection 3, 6-14] that was maintained in the Vaccination Institute,Ankara, Turkey for many years and used as the basis for vaccination ofhumans. However, due to the often severe post-vaccinal complicationsassociated with vaccinia viruses, there were several attempts togenerate a more attenuated, safer smallpox vaccine.

During the period of 1960 to 1974, Prof. Anton Mayr succeeded inattenuating CVA by over 570 continuous passages in CEF cells [Mayr etal. (1975)]. It was shown in a variety of animal models that theresulting MVA was avirulent [Mayr, A. & Danner, K. (1978), Dev. Biol.Stand. 41: 225-234]. As part of the early development of MVA as apre-smallpox vaccine, there were clinical trials using MVA-517 incombination with Lister Elstree [Stickl (1974), Prev. Med. 3: 97-101;Stickl and Hochstein-Mintzel (1971), Munich Med. Wochenschr. 113:1149-1153] in subjects at risk for adverse reactions from vaccinia. In1976, MVA derived from MVA-571 seed stock (corresponding to the 571^(st)passage) was registered in Germany as the primer vaccine in a two-stageparenteral smallpox vaccination program. Subsequently, MVA-572 was usedin approximately 120,000 Caucasian individuals, the majority childrenbetween 1 and 3 years of age, with no reported severe side effects, eventhough many of the subjects were among the population with high risk ofcomplications associated with vaccinia (Mayr et al. (1978), Zentralbl.Bacteriol. (B) 167:375-390). MVA-572 was deposited at the EuropeanCollection of Animal Cell Cultures as ECACC V94012707.

As a result of the passaging used to attenuate MVA, there are a numberof different strains or isolates, depending on the passage number in CEFcells. For example, MVA-572 was used in Germany during the smallpoxeradication program, and MVA-575 was extensively used as a veterinaryvaccine. MVA-575 was deposited on Dec. 7, 2000, at the EuropeanCollection of Animal Cell Cultures (ECACC) with the deposition numberV00120707. The attenuated CVA-virus MVA (Modified Vaccinia Virus Ankara)was obtained by serial propagation (more than 570 passages) of the CVAon primary chicken embryo fibroblasts.

Even though Mayr et al. demonstrated during the 1970s that MVA is highlyattenuated and avirulent in humans and mammals, certain investigatorshave reported that MVA is not fully attenuated in mammalian and humancell lines since residual replication might occur in these cells[Blanchard et al. (1998), J Gen Virol 79:1159-1167; Carroll & Moss(1997), Virology 238:198-211; U.S. Pat. No. 5,185,146; Ambrosini et al.(1999), J Neurosci Res 55: 569]. It is assumed that the results reportedin these publications have been obtained with various known strains ofMVA, since the viruses used essentially differ in their properties,particularly in their growth behaviour in various cell lines. Suchresidual replication is undesirable for various reasons, includingsafety concerns in connection with use in humans.

Strains of MVA having enhanced safety profiles for the development ofsafer products, such as vaccines or pharmaceuticals, have been developedby Bavarian Nordic: MVA was further passaged by Bavarian Nordic and isdesignated MVA-BN. MVA as well as MVA-BN lacks approximately 15% (31 kbfrom six regions) of the genome compared with ancestral CVA virus. Thedeletions affect a number of virulence and host range genes, as well asthe gene for Type A inclusion bodies. A sample of MVA-BN correspondingto passage 583 was deposited on Aug. 30, 2000 at the European Collectionof Cell Cultures (ECACC) under number V00083008.

MVA-BN can attach to and enter human cells where virally-encoded genesare expressed very efficiently. However, assembly and release of progenyvirus does not occur. MVA-BN is strongly adapted to primary chickenembryo fibroblast (CEF) cells and does not replicate in human cells. Inhuman cells, viral genes are expressed, and no infectious virus isproduced. MVA-BN is classified as Biosafety Level 1 organism accordingto the Centers for Disease Control and Prevention in the United States.Preparations of MVA-BN and derivatives have been administered to manytypes of animals, and to more than 2000 human subjects, includingimmune-deficient individuals. All vaccinations have proven to begenerally safe and well tolerated. Despite its high attenuation andreduced virulence, in preclinical studies MVA-BN has been shown toelicit both humoral and cellular immune responses to vaccinia and toheterologous gene products encoded by genes cloned into the MVA genome[E. Harrer et al. (2005), Antivir. Ther. 10(2):285-300; A. Cosma et al.(2003), Vaccine 22(1):21-9; M. Di Nicola et al. (2003), Hum. Gene Ther.14(14):1347-1360; M. Di Nicola et al. (2004), Clin. Cancer Res.,10(16):5381-5390].

“Derivatives” or “variants” of MVA refer to viruses exhibitingessentially the same replication characteristics as MVA as describedherein, but exhibiting differences in one or more parts of theirgenomes. MVA-BN as well as a derivative or variant of MVA-BN fails toreproductively replicate in vivo in humans and mice, even in severelyimmune suppressed mice. More specifically, MVA-BN or a derivative orvariant of MVA-BN has preferably also the capability of reproductivereplication in chicken embryo fibroblasts (CEF), but no capability ofreproductive replication in the human keratinocyte cell line HaCat[Boukamp et al (1988), J Cell Biol 106: 761-771], the human boneosteosarcoma cell line 143B (ECACC No. 91112502), the human embryokidney cell line 293 (ECACC No. 85120602), and the human cervixadenocarcinoma cell line HeLa (ATCC No. CCL-2). Additionally, aderivative or variant of MVA-BN has a virus amplification ratio at leasttwo fold less, more preferably three-fold less than MVA-575 in Helacells and HaCaT cell lines. Tests and assay for these properties of MVAvariants are described in WO 02/42480 (US 2003/0206926) and WO 03/048184(US 2006/0159699), both incorporated herein by reference.

The amplification or replication of a virus is normally expressed as theratio of virus produced from an infected cell (output) to the amountoriginally used to infect the cell in the first place (input) referredto as the “amplification ratio”. An amplification ratio of “1” definesan amplification status where the amount of virus produced from theinfected cells is the same as the amount initially used to infect thecells, meaning that the infected cells are permissive for virusinfection and reproduction. In contrast, an amplification ratio of lessthan 1, i.e., a decrease in output compared to the input level,indicates a lack of reproductive replication and therefore attenuationof the virus.

The advantages of MVA-based vaccine include their safety profile as wellas availability for large scale vaccine production. Preclinical testshave revealed that MVA-BN demonstrates superior attenuation and efficacycompared to other MVA strains (WO02/42480). An additional property ofMVA-BN strains is the ability to induce substantially the same level ofimmunity in vaccinia virus prime/vaccinia virus boost regimes whencompared to DNA-prime/vaccinia virus boost regimes.

The recombinant MVA-BN viruses, the most preferred embodiment herein,are considered to be safe because of their distinct replicationdeficiency in mammalian cells and their well-established avirulence.Furthermore, in addition to its efficacy, the feasibility of industrialscale manufacturing can be beneficial. Additionally, MVA-based vaccinescan deliver multiple heterologous antigens and allow for simultaneousinduction of humoral and cellular immunity.

In another aspect, an MVA viral strain suitable for generating therecombinant virus may be strain MVA-572, MVA-575 or any similarlyattenuated MVA strain. Also suitable may be a mutant MVA, such as thedeleted chorioallantois vaccinia virus Ankara (dCVA). A dCVA comprisesdel I, del II, del III, del IV, del V, and del VI deletion sites of theMVA genome. The sites are particularly useful for the insertion ofmultiple heterologous sequences. The dCVA can reproductively replicate(with an amplification ratio of greater than 10) in a human cell line(such as human 293, 143B, and MRC-5 cell lines), which then enable theoptimization by further mutation useful for a virus-based vaccinationstrategy (see WO 2011/092029).

DEFINITIONS

The term “antigenic determinant” refers to any molecule that stimulatesa host's immune system to make an antigen-specific immune response,whether a cellular response and/or a humoral antibody response.Antigenic determinants may include proteins, polypeptides, antigenicprotein fragments, antigens, and epitopes which still elicit an immuneresponse in a host and form part of an antigen, homologue or variant ofproteins, polypeptides, and antigenic protein fragments, antigens andepitopes including, for example, glycosylated proteins, polypeptides,antigenic protein fragments, antigens and epitopes, and nucleotidesequences encoding such molecules. Thus, proteins, polypeptides,antigenic protein fragments, antigens and epitopes are not limited toparticular native nucleotide or amino acid sequences but encompasssequences identical to the native sequence as well as modifications tothe native sequence, such as deletions, additions, insertions andsubstitutions.

Preferably, such homologues or variants have at least about 50%, atleast about 60% or 65%, at least about 70% or 75%, at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically, at leastabout 90%, 91%, 92%, 93%, or 94% and even more typically at least about95%, 96%, 97%, 98% or 99%, most typically, at least about 99% identitywith the referenced protein, polypeptide, antigenic protein fragment,antigen and epitope at the level of nucleotide or amino acid sequence.The term homologue or variant also encompasses truncated, deleted orotherwise modified nucleotide or protein sequences such as, for example,(1) RSV-F or RSV-G nucleotide sequences encoding soluble forms of thecorresponding RSV-F or RSV-G proteins lacking the signal peptide as wellas the transmembrane and/or cytoplasmic domains of the full-length RSV-For RSV-G proteins, (2) RSV-M2 or RSV-N nucleotide sequences encodingdeleted, truncated or otherwise mutated versions of the full-lengthRSV-M2 or RSV-N proteins, (3) soluble forms of the RSV-F or RSV-Gproteins lacking the signal peptide as well as the transmembrane and/orcytoplasmic domains of the full-length RSV-F or RSV-G proteins, or (4)deleted, truncated or otherwise mutated versions of the full-lengthRSV-M2 or RSV-N proteins.

Techniques for determining sequence identity between nucleic acids andamino acids are known in the art. Two or more sequences can be comparedby determining their “percent identity.” The percent identity of twosequences, whether nucleic acid or amino acid sequences, is the numberof exact matches between two aligned sequences divided by the length ofthe shorter sequences and multiplied by 100.

“Percent (%) amino acid sequence identity” with respect to proteins,polypeptides, antigenic protein fragments, antigens and epitopesdescribed herein is defined as the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe reference sequence (i.e., the protein, polypeptide, antigenicprotein fragment, antigen or epitope from which it is derived), afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forexample, using publically available computer software such as BLAST,ALIGN, or Megalign (DNASTAR) software. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximum alignment over the full length ofthe sequences being compared.

The same applies to “percent (%) nucleotide sequence identity”, mutatismutandis.

For example, an appropriate alignment for nucleic acid sequences isprovided by the local homology algorithm of Smith and Waterman, (1981),Advances in Applied Mathematics 2:482-489. This algorithm can be appliedto amino acid sequences by using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov (1986), Nucl. Acids Res.14(6):6745-6763. An exemplary implementation of this algorithm todetermine percent identity of a sequence is provided by the GeneticsComputer Group (Madison, Wis.) in the “BestFit” utility application. Thedefault parameters for this method are described in the WisconsinSequence Analysis Package Program Manual, Version 8 (1995) (availablefrom Genetics Computer Group, Madison, Wis.). A preferred method ofestablishing percent identity in the context of the present invention isto use the MPSRCH package of programs copyrighted by the University ofEdinburgh, developed by John F. Collins and Shane S. Sturrok, anddistributed by IntelliGenetics, Inc. (Mountain View, Calif.). From thissuite of packages the Smith-Waterman algorithm can be employed wheredefault parameters are used for the scoring table (for example, gap openpenalty of 12, gap extension penalty of one, and a gap of six). From thedata generated the “Match” value reflects “sequence identity.” Othersuitable programs for calculating the percent identity or similaritybetween sequences are generally known in the art, for example, anotheralignment program is BLAST, used with default parameters. For example,BLASTN and BLASTP can be used using the following default parameters:genetic code=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found at the following internet address:http://http://blast.ncbi.nlm.nih.gov/.

As used herein, a “heterologous” gene, nucleic acid, antigen, or proteinis understood to be a nucleic acid or amino acid sequence which is notpresent in the wild-type poxviral genome (e.g., MVA). The skilled personunderstands that a “heterologous gene”, when present in a poxvirus suchas MVA, is to be incorporated into the poxviral genome in such a waythat, following administration of the recombinant poxvirus to a hostcell, it is expressed as the corresponding heterologous gene product,i.e., as the “heterologous antigen” and\or “heterologous protein.”Expression is normally achieved by operatively linking the heterologousgene to regulatory elements that allow expression in thepoxvirus-infected cell. Preferably, the regulatory elements include anatural or synthetic poxviral promoter.

“Sterile immunity” as used herein means protective immunity in theabsence of detectable RSV genome when sensitive detection methods, suchas RT-qPCR, are applied.

It must be noted that, as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “an epitope” includes one ormore of epitopes and reference to “the method” includes reference toequivalent steps and methods known to those of ordinary skill in the artthat could be modified or substituted for the methods described herein.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having”. Any of the aforementioned terms (comprising, containing,including, having), though less preferred, whenever used herein in thecontext of an aspect or embodiment of the present invention can besubstituted with the term “consisting of”.

When used herein “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.

As used herein, the conjunctive term “and/or” between multiple recitedelements is understood as encompassing both individual and combinedoptions. For instance, where two elements are conjoined by “and/or”, afirst option refers to the applicability of the first element withoutthe second. A second option refers to the applicability of the secondelement without the first. A third option refers to the applicability ofthe first and second elements together. Any one of these options isunderstood to fall within the meaning, and therefore satisfy therequirement of the term “and/or” as used herein. Concurrentapplicability of more than one of the options is also understood to fallwithin the meaning, and therefore satisfy the requirement of the term“and/or.”

RSV Nucleotide Sequences and Proteins

The RSV genes as mentioned herein refer to the genes, or to a homologueor variant of the genes, encoding the corresponding protein in any RSVstrain or isolate, even though the exact sequence and/or genomiclocation of the gene may differ between strains or isolates.

Likewise, the RSV proteins mentioned herein refer to proteins, or to ahomologue or variant of the proteins, encoded and expressed by thecorresponding protein gene as defined above.

By way of example, as used interchangeably herein, the terms “F proteingene”, “F glycoprotein gene”, “RSV F protein gene”, “RSV F glycoproteingene” or “F gene” refer to the gene, or to a homologue or variant of thegene, encoding the transmembrane fusion glycoprotein in any RSV strainor isolate, even though the exact sequence and/or genomic location ofthe F protein gene may differ between strains or isolates. For example,in the A2 strain of RSV, the F(A2) protein gene comprises nucleotides5601-7499 (endpoints included) as numbered in GenBank Accession NumberM11486. The F(A2) protein gene further comprises a protein coding openreading frame (ORF) spanning nucleotides 5614-7338 (endpoints included)as numbered in GenBank Accession No. M11486. The nucleotide sequence ofthe F protein gene from RSV A2 is set forth in SEQ ID NO:28.

Also interchangeably used herein are the terms “F protein”, “Fglycoprotein”, “RSV F protein”, “RSV F glycoprotein”, or “F” which referto the heavily glycosylated transmembrane fusion glycoprotein, or to ahomologue or variant of the protein, encoded and expressed by an RSV Fprotein gene as defined above. The amino acid sequence of the F proteinfrom RSV A2 is set forth in SEQ ID NO:29. The RSV(A2) F proteincomprises a signal peptide, an extracellular domain, a transmembranedomain, and a cytoplasmic domain (see, e.g., UniProtKB/Swiss-ProtAccession No. P03420). The signal peptide of RSV A2 F protein consistsof amino acids 1-21 of SEQ ID NO:29; the extracellular domain of RSV A2F protein consists of amino acids 1-529 of SEQ ID NO:29 or amino acids22-529 of SEQ ID NO:29; the transmembrane domain of RSV A2 F proteinconsists of amino acids 530-550 of SEQ ID NO:29; and the cytoplasmicdomain of RSV A2 F protein consists of amino acids 551-574 of SEQ IDNO:29.

Likewise, also the terms “G protein gene”, “G glycoprotein gene”, “RSV Gprotein gene”, “RSV G glycoprotein gene” or “G gene” are usedinterchangeably herein. For example, in the A2 strain of RSV, the G(A2)protein gene comprises nucleotides 4626-5543 (endpoints included) asnumbered in GenBank Accession Number M11486. The G(A2) protein genefurther comprises a protein coding open reading frame (ORF) spanningnucleotides 4641-5537 (endpoints included) as numbered in GenBankAccession No. M11486. The nucleotide sequence of the G protein gene fromRSV A2 is set forth in SEQ ID NO:30.

The terms “G protein”, “G glycoprotein”, “RSV G protein”, “RSV Gglycoprotein”, or “G” refer to the heavily glycosylated transmembraneattachment glycoprotein, or to a homologue or variant of the protein.The amino acid sequence of the G protein from RSV A2 is set forth in SEQID NO:31. RSV A2 G protein comprises an extracellular domain, atransmembrane domain, and a cytoplasmic domain (see, e.g.,UniProtKB/Swiss-Prot Accession No. P03423). The extracellular domain ofRSV A2 G protein consists of amino acids 67-298 of SEQ ID NO:31; thetransmembrane domain of RSV A2 G protein consists of amino acids 38-66of SEQ ID NO:31; and the cytoplasmic domain of RSV A2 G protein consistsof amino acids 1-37 of SEQ ID NO:31.

Interchangeably used herein are also the terms “M2 protein gene”, “M2nucleocapsid protein gene”, “RSV M2 protein gene”, “RSV M2 matrixprotein gene”, “RSV M2 nucleocapsid protein gene” or “M2 gene”. Forexample, in the A2 strain of RSV, the M2(A2) protein gene comprisesnucleotides 7550-8506 (endpoints included) as numbered in GenBankAccession Number M11486. The M2(A2) protein gene further comprises aprotein coding open reading frame (ORF) spanning nucleotides 7559-8143(endpoints included) as numbered in GenBank Accession No. M11486. Thenucleotide sequence of the M2 protein gene from RSV A2 is set forth inSEQ ID NO:32.

Used interchangeably herein are the terms “M2 protein”, “M2 nucleocapsidprotein”, “RSV M2 protein”, “RSV M2 nucleocapsid protein”, “RSV M2matrix protein”, or “M2”. The amino acid sequence of the M2 protein fromRSV A2 is set forth in SEQ ID NO:33 (see, e.g., UniProtKB/Swiss-ProtAccession No. P04545).

Also, the terms “N protein gene”, “N nucleocapsid protein gene”, “RSV Nprotein gene”, “RSV N nucleocapsid protein gene” or “N gene” may be usedinterchangeably herein. For example, in the A2 strain of RSV, the N(A2)protein gene comprises nucleotides 1081-2277 (endpoints included) asnumbered in GenBank Accession Number M11486. The N(A2) protein genefurther comprises a protein coding open reading frame (ORF) spanningnucleotides 1096-2271 (endpoints included) as numbered in GenBankAccession No. M11486. The nucleotide sequence of the N protein gene fromRSV A2 is set forth in SEQ ID NO:34.

The amino acid sequence of the “N protein”, “N nucleocapsid protein”,“RSV N protein”, “RSV N nucleocapsid protein”, or “N”, terms which areinterchangeably used herein, from RSV A2 is set forth in SEQ ID NO:35(see, e.g., UniProtKB/Swiss-Prot Accession No. P03418).

CERTAIN EMBODIMENTS OF THE INVENTION

In certain embodiments, the recombinant MVA expresses at least oneheterologous nucleotide sequence encoding an antigenic determinant of anRSV membrane glycoprotein. In certain embodiments, the at least oneheterologous nucleotide sequence encoding an antigenic determinant of anRSV membrane glycoprotein encodes an RSV F antigenic determinant. Incertain embodiments, the at least one heterologous nucleotide sequenceencoding an antigenic determinant of an RSV membrane glycoproteinencodes an RSV G antigenic determinant. In certain embodiments, the RSVF antigenic determinant is derived from RSV strain A2. In certainembodiments, the RSV G antigenic determinant is derived from RSV strainA2.

In certain embodiments, the recombinant MVA comprises two heterologousnucleotide sequences, each encoding an antigenic determinant of an RSVmembrane glycoprotein. In certain embodiments, the first antigenicdeterminant of an RSV membrane glycoprotein is an RSV F antigenicdeterminant and the second antigenic determinant of an RSV membraneglycoprotein is an RSV G antigenic determinant. In certain embodiments,the RSV F antigenic determinant is derived from RSV strain A2. Incertain embodiments, the RSV G antigenic determinant is derived from RSVstrain A2. In certain embodiments, both the RSV F antigenic determinantand the RSV G antigenic determinant can be derived from RSV strain A2.

In certain embodiments, the recombinant MVA expresses at least oneheterologous nucleotide sequence encoding an antigenic determinant of anRSV membrane glycoprotein and at least one heterologous nucleotidesequence encoding an antigenic determinant of an RSV nucleocapsidprotein. In certain embodiments, the at least one heterologousnucleotide sequence encoding an antigenic determinant of an RSV membraneglycoprotein encodes an RSV F antigenic determinant and the at least oneheterologous nucleotide sequence encoding an antigenic determinant of anRSV nucleocapsid protein encodes an RSV M2 antigenic determinant. Incertain embodiments, the at least one heterologous nucleotide sequenceencoding an antigenic determinant of an RSV membrane glycoproteinencodes an RSV F antigenic determinant and the at least one heterologousnucleotide sequence encoding an antigenic determinant of an RSVnucleocapsid protein encodes an RSV N antigenic determinant. In certainembodiments, the at least one heterologous nucleotide sequence encodingan antigenic determinant of an RSV membrane glycoprotein encodes an RSVG antigenic determinant and the at least one heterologous nucleotidesequence encoding an antigenic determinant of an RSV nucleocapsidprotein encodes an RSV M2 antigenic determinant. In certain embodiments,the at least one heterologous nucleotide sequence encoding an antigenicdeterminant of an RSV membrane glycoprotein encodes an RSV G antigenicdeterminant and the at least one heterologous nucleotide sequenceencoding an antigenic determinant of an RSV nucleocapsid protein encodesan RSV N antigenic determinant.

In certain embodiments, the recombinant MVA comprises two heterologousnucleotide sequences, each encoding an antigenic determinant of an RSVmembrane glycoprotein. In certain embodiments, the first antigenicdeterminant of an RSV membrane glycoprotein is an RSV F antigenicdeterminant and the second antigenic determinant of an RSV membraneglycoprotein is an RSV G antigenic determinant. In certain embodiments,the recombinant MVA comprises two heterologous nucleotide sequences,each encoding an antigenic determinant of an RSV membrane glycoproteinand at least one heterologous nucleotide sequence encoding an antigenicdeterminant of an RSV nucleocapsid protein. In certain embodiments, thefirst antigenic determinant of an RSV membrane glycoprotein is an RSV Fantigenic determinant, the second antigenic determinant of an RSVmembrane glycoprotein is an RSV G antigenic determinant, and theantigenic determinant of an RSV nucleocapsid protein is an RSV M2antigenic determinant. In certain embodiments, the first antigenicdeterminant of an RSV membrane glycoprotein is an RSV F antigenicdeterminant, the second antigenic determinant of an RSV membraneglycoprotein is an RSV G antigenic determinant, and the antigenicdeterminant of an RSV nucleocapsid protein is an RSV N antigenicdeterminant. In certain embodiments, both the RSV F antigenicdeterminant and the RSV G antigenic determinant can be derived from RSVstrain A2.

In certain embodiments, the recombinant MVA comprises two heterologousnucleotide sequences, each encoding an antigenic determinant of an RSVmembrane glycoprotein and two heterologous nucleotide sequences, eachencoding an antigenic determinant of an RSV nucleocapsid protein. Incertain embodiments, the first antigenic determinant of an RSV membraneglycoprotein is an RSV F antigenic determinant, the second antigenicdeterminant of an RSV membrane glycoprotein is an RSV G antigenicdeterminant, the first antigenic determinant of an RSV nucleocapsidprotein is an RSV M2 antigenic determinant, and the second antigenicdeterminant of an RSV nucleocapsid protein is an RSV N antigenicdeterminant. In certain embodiments, both the RSV F antigenicdeterminant and the RSV G antigenic determinant are derived from RSVstrain A2.

In certain embodiments, the recombinant MVA comprises three heterologousnucleotide sequences, each encoding an antigenic determinant of an RSVmembrane glycoprotein and two heterologous nucleotide sequences, eachencoding an antigenic determinant of an RSV nucleocapsid protein. Incertain embodiments, the first antigenic determinant of an RSV membraneglycoprotein is an RSV F antigenic determinant and the second antigenicdeterminant of an RSV membrane glycoprotein is an RSV G antigenicdeterminant, the first antigenic determinant of an RSV nucleocapsidprotein is an RSV M2 antigenic determinant, and the second antigenicdeterminant of an RSV nucleocapsid protein is an RSV N antigenicdeterminant. In certain embodiments, both the first antigenicdeterminant of an RSV membrane glycoprotein and the second antigenicdeterminant of an RSV membrane glycoprotein are derived from RSV strainA2. In certain embodiments, the third antigenic determinant of an RSVmembrane glycoprotein is an RSV F antigenic determinant.

In certain embodiments, the recombinant MVA comprises four heterologousnucleotide sequences, each encoding an antigenic determinant of an RSVmembrane glycoprotein and two heterologous nucleotide sequences, eachencoding an antigenic determinant of an RSV nucleocapsid protein. Incertain embodiments, the first antigenic determinant of an RSV membraneglycoprotein is an RSV F antigenic determinant and the second antigenicdeterminant of an RSV membrane glycoprotein is an RSV G antigenicdeterminant, the first antigenic determinant of an RSV nucleocapsidprotein is an RSV M2 antigenic determinant, and the second antigenicdeterminant of an RSV nucleocapsid protein is an RSV N antigenicdeterminant. In certain embodiments, both the first antigenicdeterminant of an RSV membrane glycoprotein and the second antigenicdeterminant of an RSV membrane glycoprotein are derived from RSV strainA2. In certain embodiments, the third antigenic determinant of an RSVmembrane glycoprotein is an RSV F antigenic determinant. In certainembodiments, the fourth antigenic determinant of an RSV membraneglycoprotein is an RSV G antigenic determinant.

In certain embodiments, the at least one heterologous nucleotidesequence encoding an antigenic determinant of an RSV membraneglycoprotein encodes an RSV F antigenic determinant. In certainembodiments, the RSV F antigenic determinant is full-length. In certainembodiments, the RSV F antigenic determinant is truncated. In certainembodiments, the RSV F antigenic determinant is a variant RSV Fantigenic determinant. In certain embodiments, the full-length,truncated or variant RSV F antigenic determinant is derived from RSVstrain A2. In certain embodiments, the full-length RSV(A2) F antigenicdeterminant comprises the nucleotide sequence of SEQ ID NO:28 encodingthe amino acid sequence of SEQ ID NO:29. In certain embodiments, thevariant RSV(A2) F antigenic determinant comprises the nucleotidesequence of SEQ ID NO:3 encoding the amino acid sequence of SEQ ID NO:4.In certain embodiments, the truncated RSV(A2) F antigenic determinantlacks the cytoplasmic and transmembrane domains of the full-lengthRSV(A2) F antigenic determinant. In certain embodiments, the truncatedRSV(A2) F antigenic determinant comprises the nucleotide sequence of SEQID NO:15 encoding the amino acid sequence of SEQ ID NO:16. In certainembodiments, the full-length, truncated or variant RSV F antigenicdeterminant is derived from RSV strain ALong. In certain embodiments,the variant RSV(ALong) F antigenic determinant comprises the nucleotidesequence of SEQ ID NO:5 encoding the amino acid sequence of SEQ ID NO:6.In certain embodiments, the truncated RSV(ALong) F antigenic determinantlacks the cytoplasmic and transmembrane domains of the full-lengthRSV(ALong) F antigenic determinant.

In certain embodiments, the at least one heterologous nucleotidesequence encoding an antigenic determinant of an RSV membraneglycoprotein encodes an RSV G antigenic determinant. In certainembodiments, the RSV G antigenic determinant is full-length. In certainembodiments, the RSV G antigenic determinant is truncated. In certainembodiments, the RSV G antigenic determinant is a variant RSV Gantigenic determinant. In certain embodiments, the full-length,truncated or variant RSV G antigenic determinant is derived from RSVstrain A2. In certain embodiments, the full-length RSV(A2) G antigenicdeterminant comprises the nucleotide sequence of SEQ ID NO:1 encodingthe amino acid sequence of SEQ ID NO:2. In certain embodiments, thetruncated RSV(A2) G antigenic determinant lacks the cytoplasmic andtransmembrane domains of the full-length RSV(A2) G antigenicdeterminant. In certain embodiments, the full-length, truncated orvariant RSV G antigenic determinant is derived from RSV strain B. Incertain embodiments, the truncated RSV(B) G antigenic determinant lacksthe cytoplasmic and transmembrane domains of the full-length RSV(B) Gantigenic determinant. In certain embodiments, the truncated RSV(B) Gantigenic determinant comprises the nucleotide sequence of SEQ ID NO:7encoding the amino acid sequence of SEQ ID NO:8.

In certain embodiments, the at least one heterologous nucleotidesequence encoding an antigenic determinant of an RSV nucleocapsidprotein encodes an RSV M2 antigenic determinant. In certain embodiments,the RSV M2 antigenic determinant is full-length. In certain embodiments,the RSV M2 antigenic determinant is truncated. In certain embodiments,the RSV M2 antigenic determinant is a variant RSV M2 antigenicdeterminant. In certain embodiments, the full-length, truncated orvariant RSV M2 antigenic determinant is derived from RSV strain A2. Incertain embodiments, the RSV(A2) M2 antigenic determinant comprises thenucleotide sequence of SEQ ID NO:32, encoding the amino acid sequence ofSEQ ID NO:33.

In certain embodiments, the at least one heterologous nucleotidesequence encoding an antigenic determinant of an RSV nucleocapsidprotein encodes an RSV N antigenic determinant. In certain embodiments,the RSV N antigenic determinant is full-length. In certain embodiments,the RSV N antigenic determinant is truncated. In certain embodiments,the RSV N antigenic determinant is a variant RSV N antigenicdeterminant. In certain embodiments, the full-length, truncated orvariant RSV N antigenic determinant is derived from RSV strain A2. Incertain embodiments, the RSV(A2) N antigenic determinant comprises thenucleotide sequence of SEQ ID NO:34, encoding the amino acid sequence ofSEQ ID NO:35.

In certain embodiments, both the RSV N antigenic determinant and the RSVM2 antigenic determinant are encoded by a single open reading frame andseparated by a self-cleaving protease domain. In certain embodiments,the RSV M2 antigenic determinant is full-length. In certain embodiments,the RSV M2 antigenic determinant is truncated. In certain embodiments,the RSV M2 antigenic determinant is a variant RSV M2 antigenicdeterminant. In certain embodiments, the full-length, truncated orvariant RSV M2 antigenic determinant is derived from RSV strain A2. Incertain embodiments, the RSV N antigenic determinant is full-length. Incertain embodiments, the RSV N antigenic determinant is truncated. Incertain embodiments, the RSV N antigenic determinant is a variant RSV Nantigenic determinant. In certain embodiments, the full-length,truncated or variant RSV N antigenic determinant is derived from RSVstrain A2. In certain embodiments, the self-cleaving protease domain isderived from Foot and Mouth Disease Virus. In certain embodiments, theself-cleaving protease domain is the protease 2A fragment from Foot andMouth Disease Virus, comprising the nucleotide sequence of SEQ ID NO:11,encoding the amino acid sequence of SEQ ID NO:12. In certainembodiments, the at least one heterologous nucleotide sequence encodingan RSV N antigenic determinant and an RSV M2 antigenic determinantcomprises the nucleotide sequence of SEQ ID NO:17, encoding the aminoacid sequence of SEQ ID NO:18.

Integration Sites into MVA

In certain embodiments, the heterologous nucleotide sequences encodingone or more antigenic determinants of RSV membrane glycoproteins and oneor more antigenic determinants of RSV nucleocapsid proteins areincorporated in a variety of insertion sites in the MVA genome, or inthe MVA-BN genome. The heterologous nucleotide sequences encoding one ormore antigenic determinants RSV proteins can be inserted into therecombinant MVA as separate transcriptional units or as fusion genes, asdepicted in FIG. 1.

In certain embodiments, the heterologous RSV nucleotide sequences areinserted into one or more intergenic regions (IGR) of the MVA. The IGRmay be selected from IGR07/08, IGR 44/45, IGR 64/65, IGR 88/89, IGR136/137, and IGR 148/149, preferably from IGR64/65, IGR88/89, and/or IGR148/149. The heterologous RSV nucleotide sequences may be, additionallyor alternatively, inserted into one or more of the naturally occurringdeletion sites I, II, II, IV, V, or VI of the MVA. In certainembodiments, less than 5, 4, 3, or 2 of the integration sites compriseheterologous RSV nucleotide sequences.

The number of insertion sites of MVA comprising heterologous RSVnucleotide sequences can be 1, 2, 3, 4, 5, 6, 7, or more. Therecombinant MVA can comprise heterologous RSV nucleotide sequencesinserted into 4, 3, 2, or fewer insertion sites, but preferably twoinsertion sites are used. In certain embodiments, three insertion sitesare used. Preferably, the recombinant MVA comprises at least 4, 5, 6, or7 nucleotide sequences inserted into 2 or 3 insertion sites.

The recombinant MVA viruses provided herein can be generated by routinemethods known in the art. Methods to obtain recombinant poxviruses or toinsert heterologous nucleotide sequences into a poxviral genome are wellknown to the person skilled in the art. For example, methods forstandard molecular biology techniques such as cloning of DNA, DNA andRNA isolation, Western blot analysis, RT-PCR and PCR amplificationtechniques are described in Molecular Cloning, A laboratory Manual (2ndEd.) [J. Sambrook et al., Cold Spring Harbor Laboratory Press (1989)],and techniques for the handling and manipulation of viruses aredescribed in Virology Methods Manual [B. W. J. Mahy et al. (eds.),Academic Press (1996)]. Similarly, techniques and know-how for thehandling, manipulation and genetic engineering of MVA are described inMolecular Virology: A Practical Approach [A. J. Davison & R. M. Elliott(Eds.), The Practical Approach Series, IRL Press at Oxford UniversityPress, Oxford, UK (1993)(see, e.g., Chapter 9: Expression of genes byVaccinia virus vectors)] and Current Protocols in Molecular Biology[John Wiley & Son, Inc. (1998)(see, e.g., Chapter 16, Section IV:Expression of proteins in mammalian cells using vaccinia viral vector)].

For the generation of the various recombinant MVAs disclosed herein,different methods may be applicable. The nucleotide sequences to beinserted into the virus can be placed into an E. coli plasmid constructinto which DNA homologous to a section of DNA of the MVA has beeninserted. Separately, the DNA sequence to be inserted can be ligated toa promoter. The promoter-gene linkage can be positioned in the plasmidconstruct so that the promoter-gene linkage is flanked on both ends byDNA homologous to a DNA sequence flanking a region of MVA DNA containinga non-essential locus. The resulting plasmid construct can be amplifiedby propagation within E. coli bacteria and isolated. The isolatedplasmid containing the DNA gene sequence to be inserted can betransfected into a cell culture, e.g., of chicken embryo fibroblasts(CEFs), at the same time the culture is infected with MVA. Recombinationbetween homologous MVA DNA in the plasmid and the viral genome,respectively, can generate an MVA modified by the presence of foreignDNA sequences.

According to a preferred embodiment, a cell of a suitable cell cultureas, e.g., CEF cells, can be infected with a poxvirus. The infected cellcan be, subsequently, transfected with a first plasmid vector comprisinga foreign gene or genes, preferably under the transcriptional control ofa poxvirus expression control element. As explained above, the plasmidvector also comprises sequences capable of directing the insertion ofthe exogenous sequence into a selected part of the poxviral genome.Optionally, the plasmid vector also contains a cassette comprising amarker and/or selection gene operably linked to a poxviral promoter.Suitable marker or selection genes are, e.g., the genes encoding thegreen fluorescent protein, β-galactosidase,neomycin-phosphoribosyltransferase or other markers. The use ofselection or marker cassettes simplifies the identification andisolation of the generated recombinant poxvirus. However, a recombinantpoxvirus can also be identified by PCR technology. Subsequently, afurther cell can be infected with the recombinant poxvirus obtained asdescribed above and transfected with a second vector comprising a secondforeign gene or genes. In case, this gene can be introduced into adifferent insertion site of the poxviral genome, the second vector alsodiffers in the poxvirus-homologous sequences directing the integrationof the second foreign gene or genes into the genome of the poxvirus.After homologous recombination has occurred, the recombinant viruscomprising two or more foreign genes can be isolated. For introducingadditional foreign genes into the recombinant virus, the steps ofinfection and transfection can be repeated by using the recombinantvirus isolated in previous steps for infection and by using a furthervector comprising a further foreign gene or genes for transfection.

Alternatively, the steps of infection and transfection as describedabove are interchangeable, i.e., a suitable cell can at first betransfected by the plasmid vector comprising the foreign gene and, then,infected with the poxvirus. As a further alternative, it is alsopossible to introduce each foreign gene into different viruses, coinfecta cell with all the obtained recombinant viruses and screen for arecombinant including all foreign genes. A third alternative is ligationof DNA genome and foreign sequences in vitro and reconstitution of therecombined vaccinia virus DNA genome using a helper virus. A fourthalternative is homologous recombination in E. coli or another bacterialspecies between a vaccinia virus genome cloned as a bacterial artificialchromosome (BAC) and a linear foreign sequence flanked with DNAsequences homologous to sequences flanking the desired site ofintegration in the vaccinia virus genome.

Expression of RSV Genes

In one embodiment, expression of one, more, or all of the heterologousRSV nucleotide sequences is under the control of one or more poxviruspromoters. In certain embodiments, the poxvirus promoter is a Pr7.5promoter, a hybrid early/late promoter, a PrS promoter, a synthetic ornatural early or late promoter, or a cowpox virus ATI promoter. Incertain embodiments, the poxvirus promoter is selected from the groupconsisting of the PrS promoter (SEQ ID NO:39), the Pr7.5 promoter (SEQID NO:40), the PrSynIIm promoter (SEQ ID NO:41), the PrLE1 promoter (SEQID NO:42), and the PrH5m promoter (SEQ ID NO:43 [L. S. Wyatt et al.(1996), Vaccine 14(15):1451-1458]). In certain embodiments, the poxviruspromoter is the PrS promoter (SEQ ID NO:39). In certain embodiments, thepoxvirus promoter is the Pr7.5 promoter (SEQ ID NO:40). In certainembodiments, the poxvirus promoter is the PrSynIIm promoter (SEQ IDNO:41). In certain embodiments, the poxvirus promoter is the PrLE1promoter (SEQ ID NO:42). In certain embodiments, the poxvirus promoteris the PrH5m promoter (SEQ ID NO:43).

A heterologous RSV nucleotide sequence or sequences can be expressed asa single transcriptional unit. For example, a heterologous RSVnucleotide sequence can be operably linked to a vaccinia virus promoterand/or linked to a vaccinia virus transcriptional terminator. In certainembodiments, one or more heterologous RSV nucleotide sequences areexpressed as a fusion protein. The fusion protein can further comprise arecognition site for a peptidase or a heterologous self-cleaving peptidesequence. The heterologous self-cleaving peptide sequence may be the 2Apeptidase from Foot and Mouth Disease Virus.

In certain embodiments, the “transcriptional unit” is inserted by itselfinto an insertion site in the MVA genome, but may also be inserted withother transcriptional unit(s) into an insertion site in the MVA genome.The “transcriptional unit” is not naturally occurring (i.e., it isheterologous, exogenous or foreign) in the MVA genome and is capable oftranscription in infected cells.

Preferably, the recombinant MVA comprises 1, 2, 3, 4, 5, or moretranscriptional units inserted into the MVA genome. In certainembodiments, the recombinant MVA stably expresses RSV proteins encodedby 1, 2, 3, 4, 5, or more transcriptional units. In certain embodiments,the recombinant MVA comprises 2, 3, 4, 5, or more transcriptional unitsinserted into the MVA genome at 1, 2, 3, or more insertion sites in theMVA genome.

RSV Vaccines and Pharmaceutical Compositions

Since the recombinant MVA viruses, including MVA-BN, described hereinare highly replication restricted and, thus, highly attenuated, they areideal candidates for the treatment of a wide range of mammals includinghumans and even immune-compromised humans. Hence, provided herein arethe recombinant MVAs according to the present invention for use asactive pharmaceutical substances as well as pharmaceutical compositionsand vaccines, all intended for inducing an immune response in a livinganimal body, including a human.

For this, the recombinant MVA, vaccine or pharmaceutical composition canbe formulated in solution in a concentration range of 10⁴ to 10⁹TCID₅₀/ml, 10⁵ to 5×10⁸ TCID₅₀/ml, 10⁶ to 10⁸ TCID₅₀/ml, or 10⁷ to 10⁸TCID₅₀/ml. A preferred dose for humans comprises between 10⁶ to 10⁹TCID₅₀, including a dose of 10⁶ TCID₅₀, 10⁷ TCID₅₀, 10⁸ TCID₅₀ or 5×10⁸TCID₅₀.

The pharmaceutical compositions provided herein may generally includeone or more pharmaceutically acceptable and/or approved carriers,additives, antibiotics, preservatives, adjuvants, diluents and/orstabilizers. Such auxiliary substances can be water, saline, glycerol,ethanol, wetting or emulsifying agents, pH buffering substances, or thelike. Suitable carriers are typically large, slowly metabolizedmolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, lipidaggregates, or the like.

For the preparation of vaccines, the recombinant MVA viruses providedherein can be converted into a physiologically acceptable form. This canbe done based on experience in the preparation of poxvirus vaccines usedfor vaccination against smallpox as described by H. Stickl et al.,Dtsch. med. Wschr. 99:2386-2392 (1974).

For example, purified viruses can be stored at −80° C. with a titer of5×10⁸ TCID₅₀/ml formulated in about 10 mM Tris, 140 mM NaCl pH 7.7. Forthe preparation of vaccine shots, e.g., 10²-10⁸ or 10²-10⁹ particles ofthe virus can be lyophilized in 100 ml of phosphate-buffered saline(PBS) in the presence of 2% peptone and 1% human albumin in an ampoule,preferably a glass ampoule. Alternatively, the vaccine shots can beproduced by stepwise freeze-drying of the virus in a formulation. Thisformulation can contain additional additives such as mannitol, dextran,sugar, glycine, lactose or polyvinylpyrrolidone or other aids such asantioxidants or inert gas, stabilizers or recombinant proteins (e.g.,human serum albumin) suitable for in vivo administration. The glassampoule is then sealed and can be stored between 4° C. and roomtemperature for several months. However, as long as no need exists, theampoule is stored preferably at temperatures below −20° C.

For vaccination or therapy, the lyophilisate can be dissolved in anaqueous solution, preferably physiological saline or Tris buffer, andadministered either systemically or locally, i.e., parenteral,subcutaneous, intravenous, intramuscular, intranasal, or any other pathof administration known to the skilled practitioner. The mode ofadministration, the dose and the number of administrations can beoptimized by those skilled in the art in a known manner. However, mostcommonly a patient is vaccinated with a second shot about one month tosix weeks after the first vaccination shot.

Kits Comprising Recombinant MVA Viruses

Also provided herein are kits comprising any one or more of therecombinant MVAs described herein. The kit can comprise one or multiplecontainers or vials of the recombinant MVA, together with instructionsfor the administration of the recombinant MVA to a subject at risk ofRSV infection. In certain embodiments, the subject is a human. Theinstructions may indicate that the recombinant MVA is administered tothe subject in a single dose, or in multiple (i.e., 2, 3, 4, etc.)doses. In certain embodiments, the instructions indicate that therecombinant MVA virus is administered in a first (priming) and second(boosting) administration to naïve or non-naïve subjects.

Further provided is a kit comprising the recombinant MVA virus in afirst vial or container for a first administration (priming) and in asecond vial or container for a second administration (boosting). The kitmay also comprise the recombinant MVA in a third, fourth or further vialor container for a third, fourth or further administration (boosting).

Methods and Uses of Recombinant MVA Viruses

Also provided herein are methods of immunizing a subject animal, as wellas recombinant MVAs for use in methods of immunizing a subject animaland use of the recombinant MVAs provided herein in the preparation of amedicament or vaccine for immunizing a subject animal. In certainembodiments, the animal is a mammal. In certain embodiments, the mammalis a rat, rabbit, pig, mouse, or human, and the methods compriseadministering a dose of any one or more of the recombinant MVAs providedherein to the subject.

The subject is preferably a human and may be an adult, wherein the adultmay be immune-compromised. In certain embodiments, the adult is over theage of 50, 55, 60, 65, 70, 75, 80, or 85 years. In other embodiments,the subject's age is less than 5 years, less than 3 years, less than 2years, less than 15 months, less than 12 months, less than 9 months,less than 6, or less than 3 months. The subject's age may also rangefrom 0-3 months, 3-6 months, 6-9 months, 9-12 months, 1-2 years, or 2-5years.

In certain embodiments, any of the recombinant MVAs provided herein areadministered to the subject at a dose of 10⁶ to 10⁹ TCID₅₀, at a dose of10⁶ to 5×10⁸ TCID₅₀. or 10⁷ to 10⁸ TCID₅₀. The recombinant MVAs providedherein may also be administered to the subject at a dose of 10⁶, 10⁷TCID₅₀, 10⁸, or 5×10⁸ TCID₅₀. In certain embodiments, any of therecombinant MVAs provided herein are administered to a human subject ata dose of 10⁷ TCID₅₀, 10⁸, or 5×10⁸ TCID₅₀.

The recombinant MVAs provided herein are administered to the subject ina single dose, or in multiple (i.e., 2, 3, 4, etc.) doses. In certainembodiments, the recombinant MVAs are administered in a first (priming)and second (boosting) administration. In certain embodiments, the firstdose comprises 10⁷ to 10⁸ TCID₅₀ of recombinant MVA virus and the seconddose comprises 10⁷ to 10⁸ TCID₅₀ of recombinant MVA virus.

The recombinant MVAs can be administered systemically or locally,parenterally, subcutaneously, intravenously, intramuscularly, orintranasally, preferably subcutaneously or intranasally. The recombinantMVAs can also be administered by any other path of administration knownto the skilled practitioner

In another aspect, provided herein are methods of diagnosing RSVinfection and methods of determining whether a subject is at risk ofrecurrent RSV infection, which may be a severe threat, particularly fornewborn infants, children between 1 and 6 years old, and/or the elderly.

The present inventors have found that current methods of diagnosing anRSV infection may provide incorrect results. For example, an immunoassaydetecting antibodies against RSV or a viral plaque assay may notnecessarily accurately identify individuals at risk of a recurrentinfection. Indeed, the present inventors observed that even though asample taken from an individual may return a negative result in a viralplaque assay [see, e.g., W. Olszewska et al., 2004.], such results cansometimes be false negatives, since more sensitive methods sometimesdemonstrate that infectious RSV particles are still present. In fact,methods such as quantitative real time-polymerase chain reaction(qRT-PCR) are required to confirm whether a subject may actually beinfected with RSV, is at risk of recurrent infection, or indeed, whethera vaccinated subject has acquired sterile immunity to RSV. Thisdetermination may be critical, because reinfection following vaccinationsometimes causes enhanced disease, occasionally resulting in death.

Accordingly, in certain embodiments, provided herein are methods ofdetermining whether a subject is at risk of recurrent RSV infection,comprising quantitatively determining whether a sample obtained from thesubject contains RSV genomes, wherein the presence of RSV genomesindicates the likelihood of a recurrent infection with RSV. In certainembodiments, the quantitative determination of whether a sample obtainedfrom a subject contains RSV genomes is performed by qRT-PCR.

As used herein, the term “sample” refers to any biological sampleobtained from an individual, cell line, tissue culture, or other sourcecontaining polynucleotides and polypeptides or portions thereof.Biological samples include body fluids (such as, for example, blood,serum, plasma, urine, synovial fluid, spinal fluid, bronchoalveolarlavage (BAL)) and body tissues found and/or suspected to contain RSV,including clinical samples obtained, for example, from subjectsparticipating in a clinical trial or other experimental study. Methodsfor obtaining tissue biopsies and body fluids from mammals arewell-known in the art. In certain embodiments, the biological sampleincludes RSV nucleic acids.

As used interchangeably herein, the terms “RT-qPCR” or “qRT-PCR” referto a method known as “quantitative real time polymerase chain reaction”In some cases, this method may also be referred to as kinetic polymerasechain reaction (KPCR).

In certain embodiments, provided herein are methods of determiningwhether a subject has acquired sterile immunity against RSV, comprisingquantitatively determining whether a sample obtained from the subjectcontains RSV genomes, wherein the presence of RSV genomes indicates thatthe subject has not acquired sterile immunity against RSV. Also providedherein are methods of immunizing a subject that has not acquired sterileimmunity against RSV, comprising intranasally administering any one ofthe recombinant MVAs described herein to the subject. Additionally oralternatively, any one of the recombinant MVAs described herein isprovided for use in methods of immunizing a subject that has notacquired sterile immunity against RSV, the method comprisingintranasally administering any one of the recombinant MVAs describedherein to the subject. Provided herein is also the use of any of therecombinant MVAs described herein in the preparation of a medicamentand/or vaccine for immunizing a subject that has not acquired sterileimmunity against RSV, wherein the medicament or vaccine is administeredintranasally.

In certain embodiments, provided herein are methods of inducing sterileimmunity against RSV in a subject that has not acquired sterile immunityagainst RSV, comprising intranasally administering any of therecombinant MVAs described herein to the subject. Also provided hereinis any one of the recombinant MVAs described herein for use in methodsof inducing sterile immunity against RSV in a subject that has notacquired sterile immunity against RSV, the methods comprisingintranasally administering any one of the recombinant MVAs describedherein to the subject. Additionally or alternatively, provided herein isthe use of any of the recombinant MVAs described herein in thepreparation of a medicament and/or vaccine for inducing sterile immunityagainst RSV in a subject that has not acquired sterile immunity againstRSV, wherein the medicament or vaccine is administered intranasally.

Certain embodiments of the present invention also include the followingitems:

1. A recombinant modified vaccinia virus Ankara (MVA) comprising anucleotide sequence encoding an antigenic determinant of at least onerespiratory syncytial virus (RSV) membrane glycoprotein for treating orpreventing an RSV infection by intranasal administration, wherein anintramuscular administration is excluded.

2. Use of a recombinant modified vaccinia virus Ankara (MVA) comprisinga nucleotide sequence encoding an antigenic determinant of at least onerespiratory syncytial virus (RSV) membrane glycoprotein for thepreparation of a pharmaceutical composition and/or vaccine, wherein thepharmaceutical composition and/or vaccine is administered intranasallyand wherein an intramuscular administration is excluded.

3. A method of immunizing a subject, including a human, against RSVinfection, comprising intranasally administering a recombinant modifiedvaccinia virus Ankara (MVA) comprising a nucleotide sequence encoding atleast one antigenic determinant of a respiratory syncytial virus (RSV)membrane glycoprotein to the subject, including the human, wherein anintramuscular administration is excluded.

4. The recombinant MVA of item 1, the use of item 2 and/or the method ofitem 3 comprising solely intranasal administration.

5. The recombinant MVA of item 1, the use of item 2 and/or the method ofitem 3 comprising subcutaneous administration.

6. The recombinant MVA of any one of items 1 or 4 to 5, the use of anyone of items 2, 4 or 5 and/or the method of any one of items 3 to 5,wherein the recombinant MVA further comprises a nucleotide sequenceencoding an antigenic determinant of an RSV nucleocapsid protein.

7. A recombinant modified vaccinia virus Ankara (MVA) comprising atleast one nucleotide sequence encoding an antigenic determinant of arespiratory syncytial virus (RSV) membrane glycoprotein and at least onenucleotide sequence encoding an RSV nucleocapsid antigenic determinant.

8. The recombinant MVA, the use and/or method of any one of items 1 to7, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV membrane glycoprotein encodes an RSV F antigenic determinant.

9. The recombinant MVA, the use and/or method of any one of items 1 to 8further comprising at least one nucleotide sequence encoding anantigenic determinant of an RSV F membrane glycoprotein.

10. The recombinant MVA, the use and/or method of any one of items 1 to9, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV membrane glycoprotein encodes a full length RSV F membraneglycoprotein.

11. The recombinant MVA, the use and/or method of any one of items 8 to10, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV F membrane glycoprotein is derived from RSV strain A, preferablyfrom A2 and/or A_(long).

12. The recombinant MVA, the use and/or method of any one of items 8 to11, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV F membrane glycoprotein comprises a nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:4.

13. The recombinant MVA, the use and/or method of any one of items 8 to12, wherein the nucleotide sequence encoding an antigenic determinant ofan RSV F membrane glycoprotein comprises the nucleotide sequence SEQ IDNO:3.

14. The recombinant MVA, the use and/or method of any one of items 1 to13, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV membrane glycoprotein encodes a truncated RSV F membraneglycoprotein.

15. The recombinant MVA, the use and/or method of item 14, wherein thenucleotide sequence encoding the truncated RSV F membrane glycoproteinis derived from RSV strain A, preferably from A_(long).

16. The recombinant MVA, the use and/or method of item 14 or 15, whereinthe truncated RSV F membrane glycoprotein lacks the transmembranedomain.

17. The recombinant MVA, the use and/or method of any one of items 14 to16, wherein the truncated RSV F membrane glycoprotein lacks thecytoplasmic domain.

18. The recombinant MVA, the use and/or method of any one of items 8 to17, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV F membrane glycoprotein comprises a nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:6.

19. The recombinant MVA, the use and/or method of any one of items 8 to18, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV F membrane glycoprotein comprises the nucleotide sequence of SEQID NO:5.

20. The recombinant MVA, the use and/or method of any of the precedingitems, wherein the nucleotide sequence encoding an antigenic determinantof the RSV membrane glycoprotein encodes an antigenic determinant of theRSV G membrane glycoprotein.

21. The recombinant MVA, the use and/or method of any one of items 1 to20 further comprising at least one nucleotide sequence encoding anantigenic determinant of an RSV G membrane glycoprotein.

22. The recombinant MVA, the use and/or method of any one of item 1 to21, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV membrane glycoprotein encodes a full length RSV G membraneglycoprotein.

23. The recombinant MVA, the use and/or method of any one of items 20 to22, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV G membrane glycoprotein is derived from RSV strain A, preferablyfrom strain A2, and/or B.

24. The recombinant MVA, the use and/or method of any one of items 20 to23, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV G membrane glycoprotein comprises a nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:2.

25. The recombinant MVA, the use and/or method of any one of items 20 to24, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV G membrane glycoprotein comprises the nucleotide sequence SEQ IDNO:1.

26. The recombinant MVA, the use and/or method of any one of items 1 to25 wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV membrane glycoprotein encodes a truncated RSV G membraneglycoprotein.

27. The recombinant MVA, the use and/or method of item 26, wherein thenucleotide sequence encoding an antigenic determinant of a truncated RSVG membrane glycoprotein is derived from RSV strain B.

28. The recombinant MVA, the use and/or method of item 26 or 27, whereinthe truncated RSV G membrane glycoprotein lacks the transmembranedomain.

29. The recombinant MVA, the use and/or method of any one of items 26 to28, wherein the truncated RSV G membrane glycoprotein lacks thecytoplasmic domain.

30. The recombinant MVA, the use and/or method of any one of items 20 to29, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV G membrane glycoprotein comprises a nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:8.

31. The recombinant MVA, the use and/or method of any one of items 20 to30, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV G membrane glycoprotein comprises the nucleotide sequence of SEQID NO:7.

32. The recombinant MVA, the use and/or method of any one of items 6 to31, wherein the nucleotide sequence encoding an antigenic determinant ofan RSV nucleocapsid protein encodes an antigenic determinant of the RSVN nucleocapsid protein.

33. The recombinant MVA, the use and/or method of any of one of items 6to 32, wherein the nucleotide sequence encoding an antigenic determinantof an RSV nucleocapsid protein encodes an antigenic determinant of anRSV M2 matrix protein.

34. The recombinant MVA, the use and/or method of any one of items 6 to33, wherein the nucleotide sequence encoding an antigenic determinant ofan RSV nucleocapsid protein encodes a full length protein.

35. The recombinant MVA, the use and/or method of any one of items 32 to34, wherein the nucleotide sequence encoding an antigenic determinant ofthe RSV N nucleocapsid protein is derived from RSV strain A, preferablystrain A2.

36. The recombinant MVA, the use and/or method of any one of items 32 to35, wherein the nucleotide sequence encoding an antigenic determinant ofan RSV nucleocapsid protein encodes antigenic determinants of both theRSV N nucleocapsid and RSV M2 matrix proteins.

37. The recombinant MVA, the use and/or method of item 36, wherein boththe antigenic determinants of the RSV N nucleocapsid and of the RSV M2matrix proteins are encoded by a single open reading frame.

38. The recombinant MVA, the use and/or method of item 36 or 37, whereinthe antigenic determinants of the RSV N nucleocapsid and of the RSV M2matrix proteins are separated by a self-cleaving protease domain.

39. The recombinant MVA, the use and/or method of item 38, wherein theself-cleaving protease domain sequence is derived from Foot and MouthDisease Virus.

40. The recombinant MVA, the use and/or method of item 38 or 39, whereinthe self-cleaving protease domain sequence is the protease 2A fragmentsequence.

41. The recombinant MVA, the use and/or method of any one items 38 to40, wherein the self-cleaving protease domain sequence comprises anucleotide sequence encoding the amino acid sequence of SEQ ID NO:12.

42. The recombinant MVA, the use and/or method of any one of items 38 to41, wherein the self-cleaving protease domain comprises the nucleotidesequence of SEQ ID NO:11.

43. The recombinant MVA, the use and/or method of any one of items 37 to42, wherein the single open reading frame comprises a nucleotidesequence encoding the amino acid sequence of SEQ ID NO:18.

44. The recombinant MVA, the use and/or method of any one of items 37 to43, wherein the single open reading frame comprises the nucleotidesequence of SEQ ID NO:17.

45. The recombinant MVA, the use and/or method of any of the precedingitems comprising one nucleotide sequence encoding an antigenicdeterminant of an RSV membrane glycoprotein and one nucleotide sequenceencoding an antigenic determinant of an RSV nucleocapsid protein.

46. The recombinant MVA, the use and/or method of item 45 comprisingantigenic determinants of the RSV F membrane glycoprotein and of the RSVN nucleocapsid protein.

47. The recombinant MVA, the use and/or method of item 45 comprisingantigenic determinants of the RSV F membrane glycoprotein and of the RSVM2 matrix protein.

48. The recombinant MVA, the use and/or method of item 45 comprisingantigenic determinants of the RSV G membrane glycoprotein and of the RSVN nucleocapsid protein.

49. The recombinant MVA, the use and/or method of item 45 comprisingantigenic determinants of the RSV G membrane glycoprotein and of the RSVM2 matrix protein.

50. The recombinant MVA, the use and/or method of any one of items 1 to44 comprising two nucleotide sequences encoding an antigenic determinantof an RSV membrane glycoprotein and one nucleotide sequence encoding anantigenic determinant of an RSV nucleocapsid protein.

51. The recombinant MVA, the use and/or method of item 50 comprisingantigenic determinants of the RSV F and/or of the G membraneglycoproteins and of the RSV N nucleocapsid protein.

52. The recombinant MVA, the use and/or method of item 50 comprisingantigenic determinants of the RSV F and/or of the G membraneglycoproteins and of the RSV M2 matrix protein.

53. The recombinant MVA, the use and/or method of any one of items 1 to44 comprising two nucleotide sequences encoding antigenic determinantsof an RSV membrane glycoprotein and two nucleotide sequences encodingantigenic determinants of an RSV nucleocapsid protein.

54. The recombinant MVA, the use and/or method of item 53 comprisingnucleotide sequences encoding antigenic determinants of an RSV F and/orof a G membrane glycoprotein and antigenic determinants of an RSV Nnucleocapsid and/or of an M2 matrix protein.

55. The recombinant MVA, the use and/or method of any one of items 1 to44 comprising three nucleotide sequences encoding an antigenicdeterminant of an RSV membrane glycoprotein and two nucleotide sequencesencoding antigenic determinants of an RSV nucleocapsid protein.

56. The recombinant MVA, the use and/or method of item 55 comprisingantigenic determinants of two RSV F membrane glycoproteins and/or of oneRSV G membrane glycoprotein and an antigenic determinant of the RSV Nnucleocapsid protein and/or of the RSV M2 matrix protein.

57. The recombinant MVA, the use and/or method of item 55 comprisingantigenic determinants of two RSV G membrane glycoproteins and/or of oneRSV F membrane glycoprotein and an antigenic determinant of the RSV Nnucleocapsid protein and/or of the RSV M2 matrix protein.

58. The recombinant MVA, the use and/or method of any one of items 1 to44 comprising four nucleotide sequences encoding antigenic determinantsof RSV membrane glycoproteins and one nucleotide sequence encoding anantigenic determinant of an RSV nucleocapsid protein.

59. The recombinant MVA, the use and/or method of item 58 comprisingantigenic determinants of two RSV F membrane glycoproteins and/or twoRSV G membrane glycoproteins and an antigenic determinant of the RSV Nnucleocapsid protein or of the RSV M2 matrix protein.

60. The recombinant MVA, the use and/or method of any one of items 1 to44 comprising four nucleotide sequences encoding antigenic determinantsof RSV membrane glycoproteins and two nucleotide sequences encodingantigenic determinants of RSV nucleocapsid proteins.

61. The recombinant MVA, the use and/or method of item 60 comprisingantigenic determinants of two RSV F membrane glycoproteins and/or of twoRSV G membrane glycoproteins and antigenic determinants of the RSV Nnucleocapsid protein and/or of the RSV M2 matrix proteins.

62. The recombinant MVA, the use and/or method of any one of items 1 to61, wherein the MVA used for generating the recombinant MVA is MVA-BN ora derivative thereof.

63. The recombinant MVA of any one of items 1 or 4 to 62 for use as anactive pharmaceutical substance.

64. A pharmaceutical composition and/or vaccine comprising therecombinant MVA of any one of items 1 or 4 to 63 and, optionally, apharmaceutically acceptable carrier and/or diluent.

65. Use of the recombinant MVA of any one of items 1 or 4 to 63 for thepreparation of a pharmaceutical composition and/or vaccine.

66. The recombinant MVA of any one of items 6 to 63, the pharmaceuticalcomposition and/or vaccine of item 64 and/or the use of any one of items2, 4 to 6, 8 to 62 or 65 for treating or preventing an RSV infection.

67. A method of immunizing a subject, including a human, against RSVinfection, comprising administering the recombinant MVA of any one ofitems 1, 4 to 63 or 66 and/or the pharmaceutical composition and/orvaccine according to item 64 or 66 to the subject, including the human.

68. The recombinant MVA of any one of items 1, 4 to 63 or 66, thepharmaceutical composition and/or vaccine of item 64 or 66, the use ofany one of items 2, 4 to 6, 8 to 62, 65 or 66 and/or the method of anyone of items 3 to 6, 8 to 62 or 67, wherein the recombinant MVA is or isto be administered in a dose of between 10⁷-10⁹ TCID₅₀.

69. The recombinant MVA, the pharmaceutical composition and/or vaccine,the use and/or the method of any one of items 5 to 68, wherein therecombinant MVA is or is to be administered intranasally and/orsubcutaneously.

70. The recombinant MVA, the pharmaceutical composition and/or vaccine,the use and/or the method of any one of items 1 to 69, wherein therecombinant MVA is or is to be administered in a single or multipledoses to an immunologically naïve or an immunologically experiencedsubject, including a human.

71. The recombinant MVA, the pharmaceutical composition and/or vaccine,the use and/or the method of any one of items 1 to 70 for administeringto a subject, including the human, with more than 2 years of age.

72. The recombinant MVA, the pharmaceutical composition and/or vaccine,the use and/or the method of any one of items 1 to 70 for administeringto a subject, including the human, with less than 2 years of age.

73. A kit comprising one or multiple vials of the recombinant MVA of anyone of items 1, 4 to 63, 66 or 68 to 72 and instructions for theadministration of the virus to a subject at risk of RSV infection.

74. A kit comprising the recombinant MVA according to any one of items1, 4 to 63, 66 or 68 to 72 and/or the kit according to item 73,comprising the recombinant MVA in a first vial or container for a firstadministration (priming) and in a second vial or container for a secondadministration (boosting).

75. The kit according to item 73 or 74, comprising the recombinant MVAin a third, fourth or further vial or container for a third, fourth orfurther administration (boosting).

76. A cell comprising the recombinant MVA according to any one of items1, 4 to 63 or 66.

77. A method of generating a recombinant MVA according to any one ofitems 1, 4 to 63, 66 or 68 to 72, comprising the steps of:

-   -   (a) infecting a host cell with an MVA virus,    -   (b) transfecting the infected cell with a recombinant vector        comprising a nucleotide sequence encoding an RSV antigenic        determinant, said nucleotide sequence further comprising a        genomic MVA virus sequence capable of directing the integration        of the nucleotide sequence into the MVA virus genome,    -   (c) identifying, isolating and, optionally, purifying the        generated recombinant MVA virus.

78. A recombinant MVA generated according to the method of item 77.

79. A method for producing a recombinant MVA according to any one items1, 4 to 63, 66 or 68 to 72 and/or for producing an antigenic determinantexpressed from the genome of said recombinant MVA comprising the stepsof:

-   -   (a) infecting a host cell with the recombinant MVA of any one of        items 1, 4 to 63, 66 or of items 68 to 72, or transfecting the        cell with the recombinant DNA of the recombinant MVA,    -   (b) cultivating the infected or transfected cell,    -   (c) isolating the MVA and/or antigenic determinant from said        cell.

80. A recombinant MVA and/or antigenic determinant obtainable by themethod of item 79.

81. A method for determining whether a subject is at risk of recurrentRSV infection, comprising determining by means of RT-qPCR whether in asample obtained from the subject RSV is present, whereby the presence ofRSV indicates the presence of a recurrent RSV infection.

82. A method for determining whether a subject has acquired sterileimmunity against RSV, comprising determining by means of RT-qPCR whetherin a sample obtained from the subject RSV is present, whereby thepresence of RSV indicates that the subject has not acquired sterileimmunity against RSV.

83. A method of immunizing a subject diagnosed by the method of item 82to not have acquired sterile immunity against RSV, comprisingintranasally administering the recombinant MVA of any one of items 1, 4to 63, 66, 68 to 72, 78 or 80 and/or the pharmaceutical compositionand/or vaccine of any one of items 64, 66 or 68 to 72 to the subject.

84. The recombinant MVA of any one of items 1, 4 to 63, 66, 68 to 72, 78or 80 and/or the pharmaceutical composition and/or vaccine of any one ofitems 64, 66 or 68 to 72 for use in a method of immunizing a subjectdiagnosed by the method of item 82 to not have acquired sterile immunityagainst RSV, said method comprising intranasally administering saidrecombinant MVA to the subject.

85. Use of the recombinant MVA of any one of items 1, 4 to 63, 66, 68 to72, 78 or 80 for the preparation of a pharmaceutical composition and/orvaccine for immunizing a subject diagnosed by the method of item 82 tonot have acquired sterile immunity against RSV, wherein thepharmaceutical composition and/or vaccine is for intranasaladministration.

86. A method of inducing sterile immunity in a subject diagnosed by themethod of item 82 to not have acquired sterile immunity against RSV,comprising intranasally administering the recombinant MVA of any one ofitems 1, 4 to 63, 66, 68 to 72, 78 or 80 and/or the pharmaceuticalcomposition and/or vaccine of any one of items 64, 66 or 68 to 72 to thesubject.

87. The recombinant MVA of any one of items 1, 4 to 63, 66, 68 to 72, 78or 80 and/or the pharmaceutical composition and/or vaccine of any one ofitems 64, 66 or 68 to 72 for use in a method of inducing sterileimmunity in a subject diagnosed by the method of item 82 to not haveacquired sterile immunity against RSV, said method comprisingintranasally administering said recombinant MVA to the subject.

88. Use of the recombinant MVA of any one of items 1, 4 to 63, 66, 68 to72, 78 or 80 for the preparation of a pharmaceutical composition and/orvaccine for inducing sterile immunity in a subject diagnosed by themethod of item 82 to not have acquired sterile immunity against RSV,wherein the pharmaceutical composition or vaccine is for intranasaladministration.

It is to be understood that both the foregoing general and detaileddescription are exemplary and explanatory only and do not restrict orlimit the invention as claimed. The accompanying drawings, which areincorporated in and constitute a part of this specification, illustratevarious embodiments of the invention and together with the description,serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the heterologous RSV genes used in the tested recombinantMVA-constructs, MVA-mBN199B, MVA-mBN201B, MVA-mBN201BΔM2, MVA-mBN294B,MVA-mBN295B and MVA-mBN330B.

FIG. 2 shows serum RSV-specific IgG responses measured by IBLHamburg-based ELISA. Mice were immunized (s.c. or i.n.) two or threetimes with TBS or 1×10⁸ TCID₅₀ of either MVA-mBN199B, MVA-mBN201B orMVA-mBN201BΔM2. Control mice were immunized twice i.n. with 10⁶ pfu RSV.Sera were diluted 1/100 and analyzed using an RSV-specific IgG ELISAbased on the IBL Hamburg kit using plates coated with RSV F and Gproteins.

FIG. 3 shows serum RSV-specific IgG responses measured by IBLHamburg-based ELISA after serial dilution. Mice were immunized two orthree times (s.c. or i.n.) with TBS or 1×10⁸ TCID₅₀ of MVA-mBN199B,MVA-mBN201B or MVA-mBN201BΔM2. Control mice were immunized twice i.n.with 10⁶ pfu RSV. Sera were diluted (1/100, 1/200 and 1/400) andanalyzed using an RSV-specific IgG ELISA based on the IBL Hamburg kitusing plates coated with RSV F and G proteins.

FIG. 4 shows serum RSV-specific IgG responses measured by IBLHamburg-based ELISA using plates coated only with the F protein. Micewere immunized s.c. two or three times with TBS or 1×10⁸ TCID₅₀ ofMVA-mBN199B, MVA-mBN201B or MVA-mBN201BΔM2. Control mice were immunizedtwice i.n. with 10⁶ pfu RSV. Sera were diluted 1/100 and analyzed usingan RSV-specific IgG ELISA based on the IBL Hamburg kit using platescoated with RSV F protein only.

FIG. 5 shows serum RSV-specific IgG responses measured by Serion-basedELISA. Mice were immunized two or three times (s.c. or i.n.) with TBS or1×10⁸ TCID₅₀ of either MVA-mBN199B, MVA-mBN201B or MVA-mBN201BΔM2.Control mice were immunized twice i.n. with 10⁶ pfu RSV. Sera werediluted (1/100) and analyzed using an RSV-specific IgG ELISA based onthe Serion kit using plates coated with an RSV lysate.

FIG. 6 shows RSV-specific IgA versus IgG responses measured by IBLHamburg-based ELISA in bronchoalveolar lavage (BAL) fluid and sera. Micewere immunized two or three times (s.c. or i.n.) with TBS or 1×10⁸TCID₅₀ of MVA-mBN199B, MVA-mBN201B or MVA-mBN201BΔM2. Control mice wereimmunized twice i.n. with 10⁶ pfu RSV. Sera and BAL fluid were diluted(1/100) and analyzed using an RSV-specific IgG or IgA ELISA based on theIBL Hamburg kit using plates coated with RSV F and G proteins.

FIG. 7 shows RSV F-, RSV G- and RSV M2-specific T-cell responsesmeasured by ELISPOT. Mice were immunized two or three times (s.c. ori.n.) with TBS or 1×10⁸ TCID₅₀ of either MVA-mBN199B, MVA-mBN201B orMVA-mBN201BΔM2. Control mice were immunized twice i.n. with 10⁶ pfu RSV.Spleens were isolated on Day 48 and splenocytes were restimulated withthree different RSV F-specific peptides (RSV-1 (SEQ ID NO:19), RSV-2(SEQ ID NO:20) and RSV-3 (SEQ ID NO:21), one RSV G-specific peptide(RSV-4 (SEQ ID NO:22)), one RSV M2-specific peptide (RSV-9 (SEQ IDNO:27)) or MVA-BN. IFNγ-secreting cells were detected by ELISPOT. Thestimulation index was calculated as explained in the Examples.

FIG. 8 shows relative body weight loss after challenged with RSV(A2).Mice were immunized two or three times (s.c. or i.n.) with TBS or 1×10⁸TCID₅₀ of MVA-mBN199B, MVA-mBN201B or MVA-mBN201BΔM2. Control mice wereimmunized twice i.n. with 10⁶ pfu RSV. Mice were then challenged with10⁶ pfu RSV(A2) on Day 49. Weight was measured daily from the day ofchallenge. The weight on the day of challenge was used as baseline tocalculate percentage of relative body weight change.

FIG. 9 shows RSV load in lungs measured by plaque assay. Mice wereimmunized two or three times (s.c. or i.n.) with TBS or 1×10⁸ TCID₅₀ ofMVA-mBN199B, MVA-mBN201B or MVA-mBN201BΔM2. Control mice were immunizedtwice i.n. with 10⁶ pfu RSV. Mice were then challenged with 10⁶ pfuRSV(A2) on Day 49. Lungs were isolated 4 days later and the RSV load(pfu per lung) was determined by plaque assay.

FIG. 10 shows RSV load in lung measured by RT-qPCR. Mice were immunizedtwo or three times (s.c. or i.n.) with TBS or 1×10⁸ TCID₅₀ ofMVA-mBN199B, MVA-mBN201B or MVA-mBN201BΔM2. Control mice were immunizedtwice i.n. with 10⁶ pfu RSV. Mice were then challenged with 10⁶ pfu RSVA2 on Day 49. Lungs were isolated 4 days later and the RSV load(estimated based on the number of L gene copies observed) was determinedby RT-qPCR.

FIG. 11 shows IL4 level in bronchoalveolar lavage (BAL) 4 days postRSV(A2) challenge measured by ELISA. Mice were immunized two times (s.c.or i.n.) with TBS or 1×10⁸ TCID₅₀ of MVA-mBN199B or MVA-mBN201B. Controlmice were immunized twice i.n. with 10⁶ pfu RSV. Mice were thenchallenged with 10⁶ pfu RSV A2. Lungs were washed with 1 ml PBS 4 dayslater and the IL4 level in BAL was determined by ELISA. (n.d.=notdetectable)

FIG. 12 shows IL5 level in bronchoalveolar lavage (BAL) 4 days postRSV(A2) challenge measured by ELISA. Mice were immunized two times (s.c.or i.n.) with TBS or 1×10⁸ TCID₅₀ of MVA-mBN199B or MVA-mBN201B. Controlmice were immunized twice i.n. with 10⁶ pfu RSV. Mice were thenchallenged with 10⁶ pfu RSV A2. Lungs were washed with 1 ml PBS 4 dayslater and the IL5 level in BAL was determined by ELISA. (n.d.=notdetectable)

FIG. 13 shows serum RSV-specific IgG responses measured by Serion-basedELISA. Mice were immunized s.c. twice 3 weeks apart with TBS or 1×10⁸TCID₅₀ of either MVA-mBN199B, MVA-mBN201B or MVA-mBN294A. Control micewere immunized twice i.n. with 10⁶ pfu RSV. Sera of 5 mice per groupobtained 2 weeks after the last immunization were diluted and analyzedusing an RSV-specific IgG ELISA based on the Serion kit using platescoated with an RSV lysate.

FIG. 14 shows serum RSV-specific neutralizing antibody responsesmeasured by PRNT. Mice were immunized s.c. twice 3 weeks apart with TBSor 1×10⁸ TCID₅₀ of either MVA-mBN199B, MVA-mBN201B or MVA-mBN294A.Control mice were immunized twice i.n. with 10⁶ pfu RSV. Sera of 5 miceper group obtained 2 weeks after the last immunization analyzed by PRNT.

FIG. 15 shows RSV F- and RSV M2-specific T-cell responses measured byELISPOT. Mice were immunized s.c. twice 3 weeks apart with TBS or 1×10⁸TCID₅₀ of either MVA-mBN199B, MVA-mBN201B or MVA-mBN294A. Control micewere immunized twice i.n. with 10⁶ pfu RSV. Spleens were isolated on Day34 and splenocytes were restimulated with one RSV F-specific peptides(RSV-2 (SEQ ID NO:20), one RSV M2-specific peptide (RSV-9 (SEQ IDNO:27)) or MVA-BN. IFNγ-secreting cells were detected by ELISPOT. Thestimulation index was calculated as explained in the Examples.

FIG. 16 shows RSV load in lungs measured by plaque assay. Mice wereimmunized s.c. twice 3 weeks apart with TBS or 1×10⁸ TCID₅₀ of eitherMVA-mBN199B, MVA-mBN201B or MVA-mBN294A. Control mice were immunizedtwice i.n. with 10⁶ pfu RSV or i.m. with 50 μl FI-RSV. Mice were thenchallenged with 10⁶ pfu RSV(A2) on Day 49. Lungs were isolated 4 dayslater and the RSV load (pfu per lung) was determined by plaque assay.

FIG. 17 shows RSV load in lung measured by RT-qPCR. Mice were immunizeds.c. twice 3 weeks apart with TBS or 1×10⁸ TCID₅₀ of either MVA-mBN199B,MVA-mBN201B or MVA-mBN294A. Control mice were immunized twice i.n. with10⁶ pfu RSV or i.m. with 50 μl FI-RSV. Mice were then challenged with10⁶ pfu RSV A2 on Day 49. Lungs were isolated 4 days later and the RSVload (estimated based on the number of L gene copies observed) wasdetermined by RT-qPCR.

FIG. 18 shows eosinophil and neutrophil infiltrations in bronchoalveolarlavage (BAL) fluids 4 days post RSV(A2) challenge. Mice were immunizeds.c. twice 3 weeks apart with TBS or 1×108 TCID50 of either MVA-mBN199B,MVA-mBN201B or MVA-mBN294A. Control mice were immunized twice i.n. with10⁶ pfu RSV or i.m. with 50 μl FI-RSV. Mice were then challenged with10⁶ pfu RSV A2. Lungs were washed with 1 ml PBS 4 days later and thepercentage of eosinophil and neutrophil in BAL fluid was determined.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a DNA sequence encoding full-length G protein from humanRSV (hRSV) strain A2 (GenBank Accession No. M11486).

SEQ ID NO:2 is the amino acid sequence of full-length G protein fromhRSV strain A2 (GenBank Accession No. M11486).

SEQ ID NO:3 is a DNA sequence encoding full-length F protein (BNvariant) from hRSV strain A2.

SEQ ID NO:4 is the amino acid sequence of full-length F protein (BNvariant) from hRSV strain A2.

SEQ ID NO:5 is a DNA sequence encoding full-length F protein (BNvariant) from hRSV strain ALong.

SEQ ID NO:6 is the amino acid sequence encoding full-length F protein(BN variant) from hRSV strain ALong.

SEQ ID NO:7 is a DNA sequence encoding truncated G protein lacking thetransmembrane and cytoplasmic domains from hRSV strain B (GenBankAccession No. P20896).

SEQ ID NO:8 is the amino acid sequence of truncated G protein lackingthe transmembrane and cytoplasmic domains from hRSV strain B (GenBankAccession No. P20896).

SEQ ID NO:9 is a DNA sequence encoding N protein lacking a stop codonfrom hRSV strain A2 (Genbank Accession No. M11486).

SEQ ID NO:10 is the amino acid sequence of N protein lacking a stopcodon from hRSV strain A2 (Genbank Accession No. M11486).

SEQ ID NO:11 is a DNA sequence encoding a fragment of protease 2A fromFoot and Mouth Disease Virus lacking both start and stop codons.

SEQ ID NO:12 is the amino acid sequence of a fragment of protease 2Afrom Foot and Mouth Disease Virus lacking both start and stop codons.

SEQ ID NO:13 is a DNA sequence encoding full-length M2 protein lacking astart codon from hRSV strain A2 (GenBank Accession No. M11486).

SEQ ID NO:14 is the amino acid sequence encoding full-length M2 proteinlacking a start codon from hRSV strain A2 (GenBank Accession No.M11486).

SEQ ID NO:15 is a DNA sequence encoding truncated F protein lacking thetransmembrane and cytoplasmic domains (BN variant) from hRSV strain A2(GenBank Accession No. M11486).

SEQ ID NO:16 is the amino acid sequence of truncated F protein lackingthe transmembrane and cytoplasmic domains (BN variant) from hRSV strainA2 (GenBank Accession No. M11486).

SEQ ID NO:17 is a DNA sequence encoding N protein lacking a stop codonhRSV strain A2 (Genbank Accession No. M11486)+a DNA sequence encodingprotease 2A fragment from Foot and Mouth Disease Virus, lacking both astart codon and a stop codon+a DNA sequence encoding full-length M2protein lacking a start codon from hRSV strain A2 (GenBank Accession No.M11486).

SEQ ID NO:18 is the amino acid sequence of N protein from hRSV strain A2(Genbank Accession No. M11486)+the amino acid sequence of protease 2Afragment from Foot and Mouth Disease Virus, lacking a start codon+theamino acid sequence of full-length M2 protein lacking a start codon fromhRSV strain A2 (GenBank Accession No. M11486).

SEQ ID NO:19 is the amino acid sequence of RSV-1 peptide derived fromRSV F protein.

SEQ ID NO:20 is the amino acid sequence of RSV-2 peptide derived fromRSV F protein.

SEQ ID NO:21 is the amino acid sequence of RSV-3 peptide derived fromRSV F protein.

SEQ ID NO:22 is the amino acid sequence of RSV-4 peptide derived fromRSV G protein.

SEQ ID NO:23 is the amino acid sequence of RSV-5 peptide derived fromRSV G protein.

SEQ ID NO:24 is the amino acid sequence of RSV-6 peptide derived fromRSV G protein.

SEQ ID NO:25 is the amino acid sequence of RSV-7 peptide derived fromRSV G protein.

SEQ ID NO:26 is the amino acid sequence of RSV-8 peptide derived fromRSV G protein.

SEQ ID NO:27 is the amino acid sequence of RSV-9 peptide derived fromRSV M2 protein.

SEQ ID NO:28 is a DNA sequence encoding full-length F protein from hRSVstrain A2.

SEQ ID NO:29 is the amino acid sequence of full-length F protein fromhRSV strain A2.

SEQ ID NO:30 is a DNA sequence encoding full-length G protein from hRSVstrain A2.

SEQ ID NO:31 is the amino acid sequence of full-length G protein fromhRSV strain A2.

SEQ ID NO:32 is a DNA sequence encoding full-length M2 protein from hRSVstrain A2.

SEQ ID NO:33 is the amino acid sequence of full-length M2 protein fromhRSV strain A2.

SEQ ID NO:34 is a DNA sequence encoding full-length N protein from hRSVstrain A2.

SEQ ID NO:35 is the amino acid sequence of full-length N protein fromhRSV strain A2.

SEQ ID NO:36 is Primer 1 used in RT-qPCR.

SEQ ID NO:37 is Primer 2 used in RT-qPCR.

SEQ ID NO:38 is Probe 6 used in RT-qPCR.

SEQ ID NO:39 is the nucleotide sequence of the PrS promoter.

SEQ ID NO:40 is the nucleotide sequence of the Pr7.5 promoter.

SEQ ID NO:41 is the nucleotide sequence of the PrSynIIm promoter.

SEQ ID NO:42 is the nucleotide sequence of the PrLE1 promoter.

SEQ ID NO:43 is the nucleotide sequence of the PrH5m promoter.

EXAMPLES Example 1 Construction of Recombinant MVAs

Generation of recombinant MVA was done by insertion of the RSV codingsequences together with the indicated promoters (FIG. 1) into the MVAgenome via homologous recombination in CEF cells using a selectionmarker to select for recombinant MVA. The use of intergenic regions(IGRs) as insertion sites is described in WO 03/097845. In order todelete the selection marker, a second step of homologous recombinationwas employed.

MVA-BN® virus was used as starting material for the generation of therecombinant MVA-mBN199B containing the genes for RSV-A2-G andRSV-F-A2_BN in IGR88/89. The PreMaster material of MVA-mBN199 was usedas starting material for the generation of MVA-mBN201B described below.

Insertions into IGR88/89 (MVA-mBN199B):

The coding sequence for RSV-A2-G is based on the naturally occurringsequence of the RSV-A2-strain glycoprotein G. The coding sequence of thefusion protein RSV-F-A2 BN is also based on the RSV-A2 strain but wasmodified by Bavarian Nordic. Both inserted genes were synthesized byGeneart with human adapted codon usage and used for cloning of arecombination plasmid. The protein sequence of RSV-A2-G shows 100%identity to GenBank sequence P03423.1. The protein sequence of RSV-F-A2BN shows only 99% identity to GenBank sequence P03420.1 due to onesingle amino acid exchange (P to A) on position 103.

Insertions into IGR148/149 (MVA-mBN201B):

The coding sequences for RSV-N-A2 and RSV-M2-A2 are based on thenaturally occurring sequences of the respective RSV-A2-strainglycoproteins. Both genes are connected by a 2A self-cleaving peptidesequence [M. D. Ryan et al. (1991), J. Gen. Virol. 72(Pt 11):2727-2732]that allows the expression of two separate native proteins under thecontrol of a single promoter. The coding sequences for RSV-G(B) andRSV-F A long BN were truncated to remove the trans-membrane domains sothat the expressed proteins can be secreted. All inserted genes weresynthesized by Geneart with optimized codon usage and used for cloningof the recombination plasmid. The protein sequences of RSV-N-A2 andRSV-M2-A2 show 100% identity to GenBank sequence P03418.1 and P04545.1,respectively. The protein sequence of RSV-G(B) truncated shows 100%identity to GenBank sequence P20896.1. The coding sequence of RSV-F Along BN truncated was designed to contain the first 526 amino acids ofthe RSV-F protein as described by R. P. Du et al. (1994) Biotechnology(N Y) 12(8):813-818.

Deletion Mutant in M2(A2) of MVA-mBN210BΔM2:

MVA-mBN210BΔM2 includes a deletion mutation in the 12^(th) codon of theM2(A2) gene not allowing a functional M2 to be expressed. This deletioncauses the addition of the two amino acids threonine and alanine to thefirst 11 amino acids of M2 (A2) followed by a transcriptional stop (UGAstop codon).

Example 2 Immunogenicity and Efficacy of Recombinant MVA VaccinesExpressing RSV F Protein, RSV G Protein, RSV N Protein, and RSV M2Protein

Vaccine candidate MVA-mBN199B encodes the glycoprotein (G) and thefusion (F) protein of RSV, while MVA-mBN201BΔM2 and MVA-mBN201B expresstruncated versions of F and G in addition to full-length F and Gproteins, the nucleocapsid protein (N) and in case of MVA-mBN201B alsothe matrix protein (M2) of RSV (see FIG. 1). The objective of theseexperiments was to analyze the immunogenicity and protective efficacy ofMVA-mBN201BΔM2 and MVA-mBN201B compared to MVA-mBN199B after two orthree immunizations via the subcutaneous (s.c.) or intranasal (i.n.)routes of administration.

The efficacy of these constructs was tested using an RSV(A2) challengemodel in BALB/c mice. Two immunizations with MVA-mBN199B or MVA-mBN201Boffered partial protection as judged by real time quantitativepolymerase chain reaction (RT-qPCR) when applied subcutaneously and analmost complete protection when applied by the intranasal route. Theprotection offered by MVA-mBN201B was better than that offered byMVA-mBN199B. There was no difference in the humoral immune responsesinduced by the two constructs, although major differences were observedin the T-cell responses. MVA-mBN199B induced a good RSV F-specificcellular response, whereas a strong M2-specific T-cell response withMVA-mBN201B was observed, as well as a more pronounced G-specificresponse compared to MVA-mBN199B. As the IgG and T-cell responsesinduced after subcutaneous and intranasal immunization were similar, thealmost complete sterile immunity obtained by intranasal immunizationslikely correlates with the induction and secretion of RSV-specific IgAat the mucosal infection site. For MVA-mBN201BΔM2, the lack ofM2-specific T-cells responses correlated with a reduced protectioncompared to MVA-mBN201B and resulted in a similar protection thanMVA-mBN199B.

Study Design

Mice were treated subcutaneously (s.c.) or intranasally (i.n.) with1×10⁸ TCID₅₀ MVA-mBN199B (Groups 3, 4 and 5), lx 10⁸ TCID₅₀ MVA-mBN201B(Groups 6, 7 and 8), or 1×10⁸ TCID₅₀ MVA-mBN201BΔM2 (Group 9). Mice weretreated either two times (Groups 3, 4, 6, 7 and 9) or three times(Groups 5 and 8) according to Table 1. The two control groups weretreated (s.c.) twice with TBS (Group 1) or i.n. with RSV (Group 2)according to Table 7. Blood was collected on the day before immunizationor challenge as well as on the day of sacrifice. RSV-specific IgG titreswere determined by Enzyme-Linked Immunosorbent Assay (ELISA). On Day 48,half of the mice were sacrificed. Their spleens were removed andprepared for the analysis of RSV-specific T-cell responses byEnzyme-Linked Immunosorbent Spot (ELISPOT). On Day 49, the remainingmice were challenged (i.n.) with 10⁶ pfu RSV A2. Appearance and bodyweight were monitored daily starting on the day of challenge. Four dayspost challenge, mice were sacrificed by injection of an elevated dose ofKetamine-Xylazine and end-bled. After lung lavage, the lungs wereremoved and RSV load was analyzed by plaque assay and by RT-qPCR.

TABLE 1 Experimental Design Administration of Test or Reference ItemsBleed Dose and Group Schedule per ELISPOT Challenge Group Size CageInjections (Day) % Route Injection (Day) % (Day) %, # 1 5 A TBS 14 ands.c. n.a. 13, 34, 49 35 48 & 53 5 B 13, 34 — and 48& 2 5 C RSV 14 andi.n. 10⁶ pfu 13, 34, 49 35 48 and 53 5 D 13, 34 — and 48& 3 5 E MVA- 14and s.c. 1 × 10⁸ 13, 34, 49 mBN199B 35 TCID₅₀ 48 and 53 5 F 13, 34 — and48& 4 5 G MVA- 14 and i.n. 1 × 10⁸ 13, 34, 49 mBN199B 35 TCID₅₀ 48 and53 5 H 13, 34 — and 48& 5 5 J MVA- 0, 21 and s.c. 1 × 10⁸ −1, 20 34, 49mBN199B 35 TCID₅₀ 48 and 53 5 K −1, 20 34 — and 48& 6 5 L MVA- 14 ands.c. 1 × 10⁸ 13, 34, 49 mBN201B 35 TCID₅₀ 48 and 53 5 M 13, 34 — and 48&7 5 N MVA- 14 and i.n. 1 × 10⁸ 13, 34, 49 mBN201B 35 TCID₅₀ 48 and 53 5P 13, 34 — and 48& 8 5 Q MVA- 0, 21 and s.c. 1 × 10⁸ −1, 20 34, 49mBN201B 35 TCID₅₀ 48 and 53 5 R −1, 20 34 — and 48& 9 5 W MVA- 14 ands.c. 1 × 10⁸ 13, 34, 49 mBN201BΔM2 35 TCID₅₀ 48 and 53 % Relative to thefirst immunization. # Mice were challenged by the intranasal route with10⁶ pfu of RSV A2. Four days after challenge, mice were bled andsacrificed under anesthesia. BAL fluid and lungs were sampled. &On Day48, these mice were sacrificed and spleens were analyzed by ELISPOT.Study Schedule.

The schedule of the in-life phase is summarized in Table 2.

TABLE 2 Study schedule of the In-life Phase Day** Procedures −16 Arrivaland import in animal facility of 85 BALB/c mice, cage card allocationand allocation of 5 mice per cage −1 Ear clipping, inclusion/exclusionexamination of all mice −1 Pre-bleed of mice from cages J, K, Q and R(facial vein puncture right side) 0 1st administration of mice fromcages J, K, Q and R 13 pre-bleed of all mice except mice from cages J,K, Q and R (facial vein puncture right side) 14 1st administration ofall mice except mice from cages J, K, Q and R 20 Bleed of mice fromcages J, K, Q and R (facial vein puncture left side) 21 2ndadministration of mice from cages J, K, Q and R 34 Bleed of all mice(retro-bulbar vein puncture left eye) 35 2nd administration of all miceexcept mice from cages J, K, Q and R 3rd administration of mice fromcages J, K, Q and R 48 Bleed of all mice (retro-bulbar vein punctureright eye) Final bleed for cages B, D, F, H, K, M, P and R 48 Spleens ofmice from cages B, D, F, H, K, M, P and R will be removed for analysisby ELISPOT 49 Challenge of all remaining mice 49 to Appearance and bodyweight measurement daily 53 53 Final bleed, sacrifice & sampling of BAL& lung of remaining mice **Relative to the day of the 1^(st)immunization.Material and MethodsExperimental Animals.

Eighty-five female BALB/cJ Rj (H-2d) mice at the age of seven weeks wereobtained from Janvier (Route des Chênes Secs, F-53940 LeGenest-Saint-Isle, France). All mice were specific pathogen free.

Housing.

The study was performed in room 117 of the animal facility at BavarianNordic-Martinsreid. This unit was provided with filtered air at atemperature of 20-24° C. and a relative humidity between 40% and 70%.The room was artificially illuminated on a cycle of 14 hours of lightand 10 hours of darkness. The study acclimatization period was 15 days.The animals were housed in transparent SealSafe™-cages (H Temp[polysulfon] cage Type II L—Euro standard), with a floor area of 530cm². The cages were covered with an H-Temp SealSafe™ lid. The cages wereplaced in a TECNIPLAST-IVC SealSafe™ system with a SLIMLine™ circulationunit providing every single cage separately with HEPA-filtered air.Animal bedding was changed once a week.

Diet and Water.

Mice were provided with free access to irradiated maintenance diet(SSNIFF R/M-H, irradiated, V1534-727) and water (autoclaved at 121° C.for 20 minutes).

Pre-Treatment Procedures:

Identification of Animals. To individually mark animals within eachcage, ear punching was done according to standard procedures.

Inclusion/Exclusion Examination.

Inclusion/exclusion examination was done according to standardprocedures.

Blood Sampling for Pre-Bleed.

Blood Samples of approximately 150 μl were obtained by facial veinpuncture according to standard procedures. Blood samples weretransferred to the laboratory for further processing according tostandard procedures.

Treatment Procedures:

Preparation and administration of Test Items 1 to 3 and Reference Item.Preparation and administration of test and reference items was performedin a class II microbiological safety cabinet (HERAsafe®/class II type H,Kendro) according to standard procedures. Briefly, for s.c.administration, recombinant MVAs were diluted in TBS to obtain a workingsolution with a concentration of 2×10⁸ TCID₅₀/ml. 1×10⁸ TCID₅₀ in 500 μlwas injected s.c. according to standard procedures. For i.n.administration, recombinant MVAs were diluted in TBS to obtain a workingsolution with a concentration of 2×10⁹ TCID₅₀/ml. 50 μl of the dilutedviruses was administered in one nostril of anesthetized(Xylazine/Ketamine) mice according to standard procedures. 500 μl TBSwas administered s.c. according to standard procedures.

Preparation and Administration of Test Item 4/Challenge Virus.

The RSV stock vial was thawed and used as quickly as possible due tovirus instability (maximal 15 minutes on ice). Virus was kept on ice atall times and used immediately to challenge anaesthetized(Xylazine/Ketamine) mice with 100 μl of the neat virus solution by theintranasal route according to standard procedures.

Post-Treatment Procedures:

Body Weight.

Body weights were monitored on a daily basis from the day of challengeuntil sacrifice according to standard procedures.

Blood Sampling.

Blood samples (approximately 150 μl) were obtained by retro-bulbar orfacial venous puncture (for details see Table 1 and Table 2) accordingto standard procedures. Blood samples were transferred to the laboratoryfor further processing according to standard procedures.

Euthanasia.

Euthanasia of half of the mice was performed on Day 48 by cervicaldislocation. On Day 53, the remaining mice received a double dose ofKetamine-Xylazine by intra-peritoneal injection and euthanasia was doneby cutting the aorta within the peritoneal cavity.

Spleen Removal.

Spleens were removed aseptically. They were placed into tubes filledwith medium according to standard procedures. These tubes had beenimported into the animal facility and were then exported according tostandard procedures.

Lung Lavage and Lung Removal.

Bronchoalveolar lavage (BAL) fluid was collected by flushing the lungs 4times with 1 ml of PBS. The lungs were then removed and snap-frozen intwo halves in liquid nitrogen for subsequent plaque assay and RNAextraction.

Analysis: Blood Sample Processing and Storage of Sera.

Following transfer to the laboratory, the blood samples were processedto serum according to standard procedures. After preparation the serawere stored at −20° C. (±5° C.) until required for analysis.

Analysis of RSV-Specific Antibody Titres from Serum Samples.

The total RSV-specific IgG ELISA titres were determined from all serumsamples using a modified ELISA kit (Serion ELISA classic, Catalog No.ESR113G): Instead of the Alkaline Phosphatase-conjugated anti-human IgGantibody supplied with the kit, an Alkaline Phosphatase-conjugated goatanti-mouse IgG (Serotec cat: 103004) was used as the secondary antibody.

The RSV-F/G-specific IgG ELISA titers were determined from all serumsamples and BAL fluid using a modified ELISA kit (IBL-Hamburg Ref.RE56881). Instead of the POD-conjugated anti-human IgG antibody suppliedwith the kit, an HRP-conjugated sheep anti-mouse IgG (ref. BN-687-95/96,Serotec cat: AAC10P) was used as the secondary antibody.

Except for groups 4 and 7, The RSV-F-specific IgG ELISA titers weredetermined from serum samples of Day 48 using a modified ELISA kit(IBL-Hamburg Ref. RE56881 reagents and RSV (F-protein) IgG microtiterstrips Ref. RE56692). Instead of the POD-conjugated anti-human IgGantibody supplied within the kit, an HRP-conjugated sheep anti-mouse IgG(ref. BN-687-95/96 Serotec cat: AAC10P) was used as the secondaryantibody.

The RSV-specific IgA ELISA titers in sera and BAL fluid were determinedfrom Day 48 and Day 53 samples, respectively, using a modified ELISA kit(IBL-Hamburg Ref. RE56881): Instead of the POD-conjugated anti-human IgGantibody supplied within the kit, an HRP-conjugated sheep anti-mouse IgA(ref. BN-687-95/96 Serotec cat: STAR137P) was used as the secondaryantibody.

Analysis of RSV-Specific Cellular Immune Responses from Splenocytes.

The RSV F-, RSV G- and RSV M2-specific cellular responses weredetermined two weeks after the last administration by re-stimulation ofsplenocytes with specific peptides as described elsewhere (see, e.g., S.M. Varga et al. (2000); S. Johnstone et al. (2004); S. Jiang et al.,(2002); and A. B. Kulkarni et al., J. Virol. 67(7):4086-4092 (1993)) anddetection of IFNγ release from the splenocytes by ELISPOT assay.

ELISPOT Assay Method.

The Mouse IFN-Gamma-Kit (BD Biosciences, Catalog No. 551083) was usedfor the ELISPOT assay. The assay was performed according to themanufacturer's instructions. Briefly, plates were coated with thecapture antibody the day prior to splenocyte isolation. After isolation,cells were transferred to the ELISPOT plates and stimulated withdifferent peptides (see Table 3) for 20 hours at 37° C. IFNγ productionwas detected using the detection antibody. Plates were developed usingthe BD™ ELISPOT AEC Substrate Set (BD Biosciences, Catalog No. 551951)according to the manufacturer's instructions.

ELISPOT Stimulation Plan.

All conditions were tested in duplicate. RSV-1, RSV-2, RSV-3, RSV-4, andRSV-5 peptides (see Table 3) were used at a final concentration of 5μg/ml (1 μg/well) to stimulate 5×10⁵ and 2.5×10⁵ splenocytes per well.MVA (immunization control) was used at a Multiplicity of Infection (MOI)of 10 to stimulate 5×10⁵ and 2.5×10⁵ splenocytes per well andConcanavalin A (ConA [positive control]) was used at a finalconcentration of 0.5 μg/ml to stimulate 2.5×10⁵ splenocytes. As anegative control, 5×10⁵ splenocytes were cultured in medium only(RPMI-1640 supplemented with Glutamax, penicillin, streptomycin, 10%Fetal Calf Serum and 10⁵M β-mercaptoethanol.

TABLE 3 RSV-Specific Stimulation Peptide Name SpecificityPeptide Sequence RSV-1 F TYMLTNSELL (SEQ ID NO: 19) RSV-2 F KYKNAVTEL(SEQ ID NO: 20) RSV-3 F ELQLLMQSTPAANNR (SEQ ID NO: 21) RSV-4 GWAICKRIPNKKPG (SEQ ID NO: 22) RSV-5 M2 SYIGSINNI (SEQ ID NO: 27)Analysis of BAL Fluid and Lungs.

Cellular characterization of the BAL was not possible, due to stainingissues. The RSV load in the lung samples was determined by RSV plaqueassay and by RT-qPCR.

RSV Plaque Assay.

One half each of the snap-frozen lungs was homogenized in 1 ml coldmedium using a French Press (Dulbecco's Modified Eagle Mediumsupplemented with 7% Fetal Calf Serum). After a brief centrifugation,two tubes of each supernatant were titrated in two-fold serial dilutionsonto Vero cell monolayers grown in 48-well flat-bottomed plates. Sixdays later, the monolayers were washed and fixed with 1% Formaldehyde.After 24 hours, the monolayers were stained with 0.04% Neutral Red andplaques were counted.

RSV RT-qPCR.

100 μl of the homogenized lung tissue was removed immediately and RNAwas isolated using the RNeasy® Mini Kit from Qiagen (Catalog No. 74104).The reverse transcription reaction was performed using the High CapacityRNA-to-cDNA Kit from Applied Biosystems (Catalog No. 4387406). PCRspecific for the RSV L gene was performed with the following parametersin a thermal cycler: (1) 50° C. for 2 minutes; (2) 95° C. for 10minutes; (3) 45 cycles of (15 seconds at 95° C., 1 minute at 60° C.)using the Universal PCR Master Mix from Applied Biosystems (Catalog No.4352042) and a mixture of three primers: (1) primer 1 (5′-GAA CTC AGTGTA GGT AGA ATG TTT GCA-3′; SEQ ID NO:36); (2) primer 2 (5′-TTC AGC TATCAT TTT CTC TGC CAA T-3′; SEQ ID NO:37); and (3) probe 6 (5′-TTT GAA CCTGTC TGA ACA TTC CCG GTT-3′; (SEQ ID NO:38). Copy number was determinedfrom a standard curve of pMISC202 plasmid vector containing a fragmentof the RSV L gene. Similar reactions for murine beta-actin were used asinternal controls for input cDNA using a VIC/MGB-labeled probe fromApplied Biosystems (Catalog No. 4351315).

Study Documentation.

An in-life phase flow chart was prepared to collect all informationduring the individual steps of the in-life phase. In addition, mouse- orcage-specific information was recorded on the corresponding cage card.Cage cards are not considered as study raw data but a requirement fromthe Government of Upper Bavaria.

An analysis phase flow chart was prepared to collect all informationduring the individual steps of the analysis phase. Assays weredocumented in assay-specific test records or Laboratory Note Books;cross-references were documented in the analysis phase flow chart. Allassay documentation including raw data was reviewed according tostandard procedures. In addition, sample tracking sheets for serumsamples were prepared according to standard procedures.

Data Processing.

The raw data were transferred into the corresponding Excel files forfurther analysis according to standard procedures.

ELISA.

Mean values of the OD and standard errors of the mean were calculatedusing Excel.

ELISPOT.

ELISPOT plates were read with a CTL reader according to themanufacturer's instructions. The number of spot forming cells (SFC) wasdetermined for each well and transferred into an Excel file for furtherevaluation. From the incubation with 5×10⁵ and 2.5×10⁵ cells per well,the number of spots per 1×10⁶ splenocytes was calculated for each well.The mean for the negative control was calculated and was subtracted fromeach individual value prior to the calculation of the mean value permouse to obtain the Stimulation Index (SI) value (peptide-specificfrequency of IFN-γ releasing splenocytes) per mouse.

For the peptide stimulations, SI was obtained from the wells with 5×10⁵and 2.5×10⁵ cells, except when the spots were too numerous to count orfor the RSV immunized animals. In those cases only the concentration2.5×10⁵ was used. For MVA-BN stimulation, SI was obtained from the wellswith 5×10⁵, except when the spots were too numerous to count. In thatcase the concentration 2.5×10⁵ was used. Following determination of theSI for individual animals, the mean of SI (SFC per 1×10⁶ splenocytes)and standard error of the mean (SEM) was calculated per group.

Body Weight Changes.

Individual body weight values (in grams) prior to RSV challenge weretaken as baseline values. With these baseline values, individual animalbody weight changes (in %), as well as mean body weight changes of thegroups were calculated for each monitored time point post challengeusing Microsoft Excel.

RSV Plaque Assay.

The numbers of plaques were counted in the well with the three highestcountable dilutions of virus. The average number of plaques adjusted bythe dilution factor was then multiplied by 10 to obtain the titer of thesolution in pfu/ml and finally multiplied by 2 to obtain the titer perlung.

RSV RT-qPCR.

PCR amplifications were measured in real time using the ABI 7500 fromApplied Biosystems (Catalog No. 4351107) and analyzed using the SystemSoftware supplied by Applied Biosystems. All values were compared to theL gene standard and were normalized to the murine beta-actindetermination for each sample.

Results

Analysis of the Humoral Immune Response: Analysis of RSV-Specific IgGAntibody Response from Serum Samples.

Sera were first analyzed with an ELISA based on the IBL-Hamburg kitusing plates coated only with recombinant RSV F and G proteins (FIG. 2and FIG. 3). As shown in FIG. 2, similar RSV-specific IgG responses (ODsranging between 0.870 and 1.347) were observed with all three constructs(MVA-mBN199B, MVA-mBN201BΔM2 and MVA-mBN201B) after a singleimmunization and independent of the route used for immunization (s.c. ori.n.). While the second immunization resulted in a 2.0- to nearly3.5-fold increase in the antibody response (ODs ranging between 2.627 to3.407), the third s.c. injection had only a minor effect on the B-cellresponse, producing an increase of approximately 0.500 OD units comparedto ODs after the second immunization. Similar results were obtained withan ELISA based on the IBL Hamburg kit using plates coated only withrecombinant RSV F protein (FIG. 4).

After serial dilution of sera (1/100, 1/200 and 1/400) RSV F- and RSVG-specific ELISA results showed that MVA-mBN199B, MVA-mBN201BΔM2 andMVA-mBN201B induced similar RSV F- and RSV G-specific IgG responsesdespite the additional expression of a truncated RSV F protein and atruncated RSV G protein by MVA-mBN201BΔM2 and MVA-mBN201B (FIG. 3).After two s.c. immunizations with the constructs, the B-cell responsewas still lower compared to the immunization with RSV alone (positivecontrol). To reach the level of antibody response induced by two i.n.applications of RSV required a third s.c. immunization with the MVA-mBNconstructs. In contrast, 2 i.n. immunizations with MVA-mBN199B andMVA-mBN201B induced similar B-cell responses as two immunizations withRSV alone or 3 s.c. immunizations with MVA-mBN199B, MVA-mBN201BΔM2 andMVA-mBN201B, when analyzed with ELISA based on the IBL Hamburg kit (FIG.3).

When sera were analyzed again by ELISA based on the Serion kit usingplates coated with an RSV lysate, again no differences betweenMVA-mBN199B, MVA-mBN201BΔM2 and MVA-mBN201B were found. No differencesbetween 2 and 3 immunizations, or between the s.c. and i.n. routes ofadministration were observed either. In addition, the responses were alllower than the antibody response induced by 2 i.n. applications of RSV(FIG. 5).

Analysis of RSV-Specific IgA Antibody Responses.

RSV F- and RSV G-specific IgA (based on the IBL Hamburg kit) wasmeasured in BAL fluid 4 days post-challenge (Day 53). In addition, alsoBAL and sera for RSV F- and RSV G-specific IgG by ELISA were analyzed.Results were compared to the results obtained in sera just beforechallenge (Day 48) and are shown in FIG. 6.

As expected, IgA responses were detected only after i.n. applicationwith RSV, MVA-mBN199B and MVA-mBN201B. Although IgG could also bedetected in the BAL, IgA was detected at a higher level after i.n.application. Serum levels of IgA were much lower than IgG levelsindependent of the route of application.

Analysis of RSV-Specific Cellular Immune Responses.

T-cell responses were analyzed in the spleen by ELISPOT two weeks afterthe last immunization (FIG. 7). MVA-mBN199B administered by the i.n. ors.c. route induced a strong RSV-F specific T-cell response. This immuneresponse was mainly directed against the RSV-F-specific peptide RSV-2,which is immunodominant in the absence of RSV-M2. The response wasaround 2000 spots per 10⁶ splenocytes after the 2^(nd) s.c.immunization, and around 4000 after the 3^(rd) s.c. injection or 2^(nd)intranasal application. Similar to the response to RSV intranasalapplications, a low G-specific response to peptide RSV-4 was detectedafter immunization with MVA-mBN199B (approximately 500 spots per 10⁶splenocytes) and as expected, MVA-mBN199B did not induce M2-specificT-cells. The M2 peptide is the immuno-dominant peptide of RSV in mice.Consequently, RSV intranasal immunizations induced a good M2-specificT-cell response above 1000 spots per 10⁶ splenocytes and almost noF-specific T-cell response.

Like MVA-mBN199B, MVA-mBN201B induced a strong T-cell response, but itwas dominated by M2 (above 4000 spots per 10⁶ splenocytes independent ofthe number of doses administered or the route of administration). Eventhe G-specific response induced by MVA-mBN201B was at least 3-foldhigher than the G-specific response induced by MVA-mBN199B or RSV. Incontrast to MVA-mBN199B, the F-specific response induced by MVA-mBN201Bwas much lower, with less than 600 spots per 10⁶ splenocytes for theRSV-2 peptide.

RSV Challenge with RSV A2 Strain.

Mice were challenged intranasally with 10⁶ pfu of RSV(A2) two weeksafter the last immunization. Body weight was monitored daily. Four dayspost challenge, mice were sacrificed. After lung lavage with 1 ml PBS,lungs were removed and the RSV load in lung was determined by plaqueassay and RT-qPCR conducted as described above.

Body Weight Changes.

All mice lost weight one day post-challenge, most probably due toanesthesia or the i.n. challenge itself (FIG. 8). TBS-treated micestarted to significantly lose weight 4 days post-RSV challenge. Incontrast, mice that received RSV intranasally for the third time did notshow body weight loss. All mice immunized s.c. with MVA-mBN199B,MVA-mBN201B or MVA-mBN201BΔM2 lost about 20% weight 4 days postchallenge. Such weight loss was described earlier by Olszewska et al.(Vaccine 23:215 (2004)). However our RT-qPCR results (FIG. 10) show thatit correlates to a better protection and earlier elimination of RSV fromlung via the vaccine-primed CTL response compared to the normalclearance of primary RSV infection. When applied i.n., MVA-mBN201Bimmunized mice had a similar weight loss than s.c. immunized mice 2 dayspost-challenge, but had recovered 4 days post-challenge due to the lowRSV load in lungs compared to the s.c. route (FIG. 10). Like theRSV-immunized group, mice immunized i.n. with MVA-mBN199B showed noweight loss.

RSV Load Measured by Plaque Assay.

Four days post challenge an average of 57671 pfu per lung for thenon-immunized mice was detected (FIG. 9). As in the RSV-immunizedcontrol group, no RSV A2 plaques were detected in the lungs of animalsimmunized with MVA-mBN199B, MVA-mBN201B or MVA-mBN201BΔM2 after 2 s.c.or i.n. applications.

RSV Load Measured by Quantitative Real-Time PCR.

The RSV load in lung was also analyzed by RT-qPCR (FIG. 10). While RSVwas not detected by plaque assay in any of the vaccinated mice, RSVgenomes were still detectable in mice immunized three times withMVA-mBN199B. After 3 immunizations with MVA-mBN199B, the RSV load was 38times lower compared to the TBS control group. RSV genomes were alsodetectable after three immunizations with MVA-mBN201B but the load was158 times lower compared to the TBS control group. There was no majordifference between mice immunized two or three times. Interestingly, thedecrease in the RSV load observed with MVA-mBN201B was not observedafter vaccination with MVA-mBN201BΔM2 which was in absence of M2equivalent to MVA-mBN199B.

Nearly complete protection comparable that obtained in the group treatedwith RSV was observed after i.n. immunization with MVA-mBN201B, althougha few copies of the L gene were still detectable in one mouse out offive. Intranasal immunization with MVA-mBN199B also induced a strongdecrease in the RSV load, but the L gene was still detected at a lowlevel in three mice out of four.

DISCUSSION AND CONCLUSIONS

Although MVA-mBN201B expresses truncated versions of RSV F and Gproteins in addition to the full-length RSV F and G proteins alsoincluded in the MVA-mBN199B construct, MVA-mBN201B induced a humoralimmune response of similar magnitude. Both constructs induced anantibody response directed mostly against the RSV F protein as judged bysimilarly good responses measured in the RSV F-only ELISA compared tothe RSV F and G ELISA. The antibody level following two i.n.applications was higher than after two s.c. applications. A third s.c.application was required to reach the antibody response level induced bytwo i.n. applications. In contrast, no major differences were observedin the T-cell responses induced using the s.c. versus i.n. routes, orusing 2 versus 3 s.c. applications. However, MVA-mBN199B induced a goodRSV F-specific cellular response, whereas a strong M2-specific T-cellresponse with MVA-mBN201B was observed. The RSV G-specific responseinduced by MVA-mBN201B was also more pronounced compared to MVA-mBN199B.The pattern of T-cell response induced by MVA-mBN201B was similar to theT-cell response induced by RSV immunization, albeit much higher.

Independent of the routes or the number of applications, both constructsprotected mice from challenge with RSV(A2), and no replicating viruscould be recovered from the lungs. However, as previously observed, s.c.immunizations with MVA-mBN199B or MVA-mBN201B did not result in sterileimmunity (i.e., immunity which persists even after the targetedinfectious agent is cleared from the body). The genomic RSV load(measured by levels of the viral RNA polymerase (L) gene) in the lungsof mice immunized by s.c. application of MVA-mBN199B or MVA-mBN201B wassignificantly reduced but still detectable by quantitative RT-PCR, and athird s.c. immunization had no beneficial impact on viral load despiteits increase in RSV-specific IgG levels. The reduction in RSV L proteinexpression was a little more pronounced after vaccination withMVA-mBN201B compared to MVA-mBN199B, which might be due to the increasedM2-specific CD8+ T-cell response, as the RSV genomic load was higher inanimals vaccinated with MVA-mBN201BΔM2 than for animals vaccinated withMVA-mBN201B, like MVA-mBN199B.

Sterile immunity was almost obtained after two i.n. applications ofMVA-mBN199B or MVA-mBN201B. This observation correlated with theinduction and secretion of RSV-specific IgA at the mucosal infectionsite.

Example 3 Safety of Recombinant MVA Vaccines Expressing RSV F Protein,RSV G Protein, RSV N Protein, and RSV M2 Protein, Compared to FI-RSV

Vaccine candidate MVA-mBN199B encodes the glycoprotein (G) and thefusion (F) protein of RSV, while MVA-mBN201B express truncated versionsof F and G in addition to full-length proteins, the nucleocapsid protein(N) and the matrix protein (M2) of RSV (see FIG. 1). The objective ofthese experiments was to analyze the safety of MVA-mBN199B andMVA-mBN201B compared to FI-RSV after two immunizations via thesubcutaneous (s.c.) or intranasal (i.n.) routes of administration.

The safety of these constructs was tested using an RSV(A2) challengemodel in BALB/c mice. Two immunizations with MVA-mBN199B or MVA-mBN201Bdid not induced increased IL4 and IL5 secretion in BAL following RSV(A2)challenge, compared to FI-RSV.

Study Design

Mice were treated twice three weeks apart subcutaneously (s.c.) orintranasally (i.n.) with 1×10⁸ TCID₅₀ MVA-mBN199B (Groups 3 and 4),1×10⁸ TCID₅₀ MVA-mBN201B (Groups 5 and 6) according to Table x. Thethree control groups were treated (s.c.) twice with TBS (Group 1) ori.n. with RSV (Group 2) or i.m. with 30 μg FI-RSV(Group 7), according toTable x. On Day 35, mice were challenged (i.n.) with 10⁶ pfu RSV A2.Four days post challenge, mice were sacrificed by injection of anelevated dose of Ketamine-Xylazine and end-bled. After lung lavage, IL4and IL5 level were analyzed in BAL by ELISA.

TABLE 4 Experimental Design Administration of Test or Reference ItemsDose Group Schedule per Challenge Group Size Injections (Day) % RouteInjection (Day) %, # 1 5 TBS 0 and 21 s.c. n.a. 35 2 5 RSV i.n. 10⁶ pfu3 5 MVA- s.c. 1 × 10⁸ mBN199B TCID₅₀ 4 5 i.n. 1 × 10⁸ TCID₅₀ 5 5 MVA-s.c. 1 × 10⁸ mBN201B TCID₅₀ 6 5 i.n. 1 × 10⁸ TCID₅₀ 7 5 FI-RSV i.m. 30μg % Relative to the first immunization. # Mice were challenged by theintranasal route with 10⁶ pfu of RSV A2. Four days after challenge, micewere bled and sacrificed under anesthesia. BAL fluid were sampled.Study Schedule.

The schedule of the in-life phase is summarized in Table y.

TABLE 5 Study schedule of the In-life Phase Day** Procedures −9 Arrivaland import in animal facility of BALB/c mice, cage card allocation andallocation of 5 mice per cage −1 Ear clipping, inclusion/exclusionexamination of all mice 0 1^(st) administration of mice 21 2^(nd)administration of mice 35 RSV(A2) Challenge 39 Final bleed, sacrifice &sampling of BAL **Relative to the day of the 1^(st) immunization.Material and MethodsExperimental Animals.

female BALB/cJ Rj (H-2d) mice at the age of seven weeks were obtainedfrom Janvier (Route des Chênes Secs, F-53940 Le Genest-Saint-Isle,France). All mice were specific pathogen free.

Housing.

The study was performed in room 117 of the animal facility at BavarianNordic-Martinsreid. This unit was provided with filtered air at atemperature of 20-24° C. and a relative humidity between 40% and 70%.The room was artificially illuminated on a cycle of 14 hours of lightand 10 hours of darkness. The study acclimatization period was 15 days.The animals were housed in transparent SealSafe™-cages (H Temp[polysulfon] cage Type II L—Euro standard), with a floor area of 530cm². The cages were covered with an H-Temp SealSafe™ lid. The cages wereplaced in a TECNIPLAST-IVC SealSafe™ system with a SLIMLine™ circulationunit providing every single cage separately with HEPA-filtered air.Animal bedding was changed once a week.

Diet and Water.

Mice were provided with free access to irradiated maintenance diet(SSNIFF R/M-H, irradiated, V1534-727) and water (autoclaved at 121° C.for 20 minutes).

Pre-Treatment Procedures:

Identification of Animals. To individually mark animals within eachcage, ear punching was done according to standard procedures.

Inclusion/Exclusion Examination.

Inclusion/exclusion examination was done according to standardprocedures.

Blood Sampling for Pre-Bleed.

Blood samples of approximately 150 μl were obtained by facial veinpuncture according to standard procedures. Blood samples weretransferred to the laboratory for further processing according tostandard procedures.

Treatment Procedures:

Preparation and administration of Test Items and Reference Item.Preparation and administration of test and reference items was performedin a class II microbiological safety cabinet (HERAsafe®/class II type H,Kendro) according to standard procedures. Briefly, for s.c.administration, recombinant MVAs were diluted in TBS to obtain a workingsolution with a concentration of 2×10⁸ TCID₅₀/ml. 1×10⁸ TCID₅₀ in 500 μlwas injected s.c. according to standard procedures. For i.n.administration, recombinant MVAs were diluted in TBS to obtain a workingsolution with a concentration of 2×10⁹ TCID₅₀/ml. 50 μl of the dilutedviruses was administered in one nostril of anesthetized(Xylazine/Ketamine) mice according to standard procedures. 500 μl TBSwas administered s.c. according to standard procedures.

Preparation and Administration of RSV(A2) Virus.

The RSV stock vial was thawed and used as quickly as possible due tovirus instability (maximal 15 minutes on ice). Virus was kept on ice atall times and used immediately to challenge anaesthetized(Xylazine/Ketamine) mice with 100 μl of the neat virus solution by theintranasal route according to standard procedures.

Preparation and Administration of FI-RSV.

30 μg FI-RSV in 40 μl was injected intramuscularly.

Euthanasia.

On Day 35, the remaining mice received a double dose ofKetamine-Xylazine by intra-peritoneal injection and euthanasia was doneby cutting the aorta within the peritoneal cavity.

Lung Lavage.

Bronchoalveolar lavage (BAL) fluid was collected by flushing the lungs 4times with 1 ml of PBS.

Analysis

IL-4 and IL-5 levels were measured in bronchoalveolar lavage (BAL)supernatant using commercially available ELISA kits (mIL4 PLATINUM ELISAfrom eBIOSCIENCE Cat N^(o) BMS613 and READY-SET-GO MIL-5 ELISA fromeBIOSCIENCE Cat N^(o) 88-7054-22).

Study Documentation.

An in-life phase flow chart was prepared to collect all informationduring the individual steps of the in-life phase. In addition, mouse- orcage-specific information was recorded on the corresponding cage card.Cage cards are not considered as study raw data but a requirement fromthe Government of Upper Bavaria.

An analysis phase flow chart was prepared to collect all informationduring the individual steps of the analysis phase. Assays weredocumented in assay-specific test records or Laboratory Note Books;cross-references were documented in the analysis phase flow chart. Allassay documentation including raw data was reviewed according tostandard procedures. In addition, sample tracking sheets for serumsamples were prepared according to standard procedures.

Data Processing.

The raw data were transferred into the corresponding Excel files forfurther analysis according to standard procedures.

ELISA.

Cytokine concentrations were determined from the standard curve of therespective ELISA kits.

Results

An increase of IL-4 (FIG. 11) and IL-5 (FIG. 12) production like thatobserved with FI-RSV was not observed for MVA-mBN199B or MVA-mBN201B.Both cytokines were below the detection level when mice were immunizedi.n. with MVA-mBN199B or MVA-mBN201B.

DISCUSSION AND CONCLUSIONS

Both MVA-mBN199B and MVA-mBN201B do not induce enhanced disease comparedto FI-RSV as assessed by TH2 response.

Example 4 Comparison of Immunogenicity Efficacy and Safety of DifferentRecombinant MVA Vaccines Expressing RSV F Protein, RSV G Protein, RSV NProtein, and RSV M2 Proteins

Vaccine candidate MVA-mBN199B encodes the glycoprotein (G) and thefusion (F) protein of RSV, MVA-mBN201B expresses truncated versions of Fand G in addition to full-length proteins, the nucleocapsid protein (N)and the matrix protein (M2) of RSV and MVA-mBN294B expresses one F and 2G full-length proteins, the nucleocapsid protein (N) and the matrixprotein (M2) of RSV (see FIG. 1). MVA-mBN294A is in an intermediateproduct in the cloning MVA-mBN294B which still has one cloning cassettein. This cloning cassette does not impact either transgene expression orthe immunogenic properties of the transgenic proteins. The objective ofthis experiment was to analyze the Immunogenicity, efficacy and safetyof MVA-mBN294A compared to MVA-mBN199B and MVA-mBN201B after twoimmunizations via the subcutaneous (s.c.) route of administration.

The immunogenicity efficacy and safety of these constructs was testedusing an RSV(A2) challenge model in BALB/c mice. We confirmed thatdespite the changes in MVA-mBN294A (equivalent to MVA-mBN294B) comparedto MVA-mBN201B, it induced similar B- and T-cell responses and offeredsimilar protection. This experiment showed that any constructs(MVA-mBN201B or MVA-mBN294A) expressing at least one antigenicdeterminant of an RSV membrane glycoprotein (F or G) and at least oneantigenic determinant of an RSV nucleocapsid protein (N or M2) inducesbetter protection than a construct expressing only antigenicdeterminants of RSV membrane glycoproteins (MVA-mBN199B)

Study Design

Mice were vaccinated (s.c.) with 1×10⁸ TCID₅₀ MVA-mBN294A, MVA-mBN199Bor MVA-mBN201B in a prime-boost schedule (Day 0 and 21) according toTable 6. The control groups were treated twice subcutaneously with TBSor with RSV-A2 according to Table 6. Formalin Inactivated (FI)-RSV wasinjected intramuscularly (i.m.) either once or twice according to Table6.

Blood was collected one day prior to each immunization and prior tochallenge, as well as on the day of sacrifice. For 5 animals of groups 1to 5 on Day 34, RSV-specific IgG titers and RSV-specific neutralizingantibody titers were determined by ELISA and PRNT respectively.

On Day 34, some mice (Table 6) were sacrificed by injection of a lethaldose of ketamine-xylazine and final bleed. Spleens were removed andprepared for the analysis of RSV-specific T cell responses by ELISPOT.

On Day 35, the remaining mice (Table 6) were challenged with 10⁶ pfuRSV-A2. Four days post-challenge, mice were sacrificed by injection of alethal dose of ketamine-xylazine and final bleed. After lung lavage, thelungs were removed and RSV load was analyzed by plaque assay andRT-qPCR. Cellular infiltration and cytokines level in Bonchoalveolarlavage (BAL) fluids were analyzed.

TABLE 6 Experimental Design Administration of Test or Reference ItemsSchedule for Dose Group Injection per Bleed Challenge Group SizeInjections (Day)¹ Route Injection (Day)¹ (Day)^(1,2) 1 10 TBS 0 and 21s.c. — −1, 20, 34 and 35 39 5 −1, 20 and 34^(&) — 2 10 RSV i.n. 10⁶ pfu−1, 20, 34 and 35 39 5 −1, 20 and 34^(&) — 3 10 MVA- s.c. 1 × 10⁸ −1,20, 34 and 35 mBN199B TCID₅₀ 39 5 −1, 20 and 34^(&) — 4 10 MVA- −1, 20,34 and 35 mBN201B 39 5 −1, 20 and 34^(&) — 5 10 MVA- −1, 20, 34 and 35mBN294A 39 5 −1, 20 and 34^(&) — 6 10 FI-RSV i.m. 50 μl −1, 20, 34 and35 39 5 −1, 20 and 34^(&) — 7 5 FI-RSV 0 i.m. 50 μl −1, 20, 34 and 35 39¹relative to the first immunization ²Mice will be challenged by theintranasal route with 10⁶ pfu of RSV-A2. Four days after challenge, micewill be bled, sacrificed under anesthesia and BAL and lungs will besampled ^(&)on Day 34, these mice will be sacrificed and spleens will beanalyzed by ELISPOTStudy Schedule.

The schedule of the in-life phase is summarized in Table 7.

TABLE 7 Study schedule of the Part 1 of the In-life Phase Day¹Procedures −9 Arrival and import in animal facility of BALB/c mice, cagecard allocation and allocation of 5 mice per cage −1 Ear clipping,inclusion/exclusion examination of all mice −1 Pre-bleed of all mice(facial vein puncture right side) 0 1^(st) administration 20 Bleed ofall mice (facial vein puncture left side) 21 2^(nd) administration 34Final bleed, sacrifice and sampling of spleen for cages B, D, F, H, Kand M 34 Bleed of all remaining mice (retro-bulbar vein puncture righteye) 35 Challenge of all remaining mice 35 to 39 Appearance and bodyweight measurement daily 39 Final bleed, sacrifice and sampling of BAFand lung of mice ¹relative to the day of the 1^(st) immunizationMaterial and MethodsExperimental Animals.

female BALB/cJ Rj (H-2d) mice at the age of seven weeks were obtainedfrom Janvier (Route des Chênes Secs, F-53940 Le Genest-Saint-Isle,France). All mice were specific pathogen free.

Housing.

The study was performed in room 117 of the animal facility at BavarianNordic-Martinsreid. This unit was provided with filtered air at atemperature of 20-24° C. and a relative humidity between 40% and 70%.The room was artificially illuminated on a cycle of 14 hours of lightand 10 hours of darkness. The study acclimatization period was 15 days.The animals were housed in transparent SealSafe™-cages (H Temp[polysulfon] cage Type II L—Euro standard), with a floor area of 530cm². The cages were covered with an H-Temp SealSafe™ lid. The cages wereplaced in a TECNIPLAST-IVC SealSafe™ system with a SLIMLine™ circulationunit providing every single cage separately with HEPA-filtered air.Animal bedding was changed once a week.

Diet and Water.

Mice were provided with free access to irradiated maintenance diet(SSNIFF R/M-H, irradiated, V1534-727) and water (autoclaved at 121° C.for 20 minutes).

Pre-Treatment Procedures:

Identification of Animals.

To individually mark animals within each cage, ear punching was doneaccording to standard procedures.

Inclusion/Exclusion Examination.

Inclusion/exclusion examination was done according to standardprocedures.

Blood Sampling for Pre-Bleed.

Blood samples of approximately 150 μl were obtained by facial veinpuncture according to standard procedures. Blood samples weretransferred to the laboratory for further processing according tostandard procedures.

Treatment Procedures:

Preparation and administration of Test Items and Reference Item.Preparation and administration of test and reference items was performedin a class II microbiological safety cabinet (HERAsafe®/class II type H,Kendro) according to standard procedures. Briefly, for s.c.administration, recombinant MVAs were diluted in TBS to obtain a workingsolution with a concentration of 2×10⁸ TCID₅₀/ml. 1×10⁸ TCID₅₀ in 500 μlwas injected s.c. according to standard procedures. 500 μl TBS wasadministered s.c. according to standard procedures.

Preparation and Administration of RSV(A2) Virus.

The RSV stock vial was thawed and used as quickly as possible due tovirus instability (maximal 15 minutes on ice). Virus was kept on ice atall times and used immediately to challenge anaesthetized(Xylazine/Ketamine) mice with 100 μl of the neat virus solution by theintranasal route according to standard procedures.

Preparation and Administration of FI-RSV:

50 μl of FI-RSV was applied i.m.

Post-Treatment Procedures:

Blood Sampling.

Blood samples (approximately 150 μl) were obtained by retro-bulbar orfacial venous puncture (for details see Table 7) according to standardprocedures. Blood samples were transferred to the laboratory for furtherprocessing according to standard procedures.

Euthanasia.

Mice received a double dose of Ketamine-Xylazine by intra-peritonealinjection and euthanasia was done by cutting the aorta within theperitoneal cavity.

Spleen Removal.

Spleens were removed aseptically. They were placed into tubes filledwith medium according to standard procedures. These tubes had beenimported into the animal facility and were then exported according tostandard procedures.

Lung Lavage and Lung Removal.

Bronchoalveolar lavage (BAL) fluid was collected by flushing the lungs 4times with 1 ml of PBS. The lungs were then removed and snap-frozen intwo halves in liquid nitrogen for subsequent plaque assay and RNAextraction.

Analysis:

Blood Sample Processing and Storage of Sera.

Following transfer to the laboratory, the blood samples were processedto serum according to standard procedures. After preparation the serawere stored at −20° C. (±5° C.) until required for analysis.

Analysis of RSV-Specific Antibody Titres from Serum Samples.

The total RSV-specific IgG ELISA titres were determined from all serumsamples using a modified ELISA kit (Serion ELISA classic, Catalog No.ESR113G): Instead of the Alkaline Phosphatase-conjugated anti-human IgGantibody supplied with the kit, an Alkaline Phosphatase-conjugated goatanti-mouse IgG (Serotec cat: 103004) was used as the secondary antibody.

Analysis of RSV-Specific neutralizing Antibody Titres from SerumSamples.

Briefly, 2-fold serial dilutions of the test sera were prepared and adefined number of RSV plaque forming units (pfu) were added to the serumdilution. After 185 min incubation at 36° C. (±2° C.) and 5% CO₂ (±1%).it was added to pre-seeded plates containing Vero cells. Two days laterplates were fixed, immuno-stained with a mixture of RSV-specificantibodies and plaques were counted.

Analysis of RSV-Specific Cellular Immune Responses from Splenocytes.

The RSV F- and RSV M2-specific cellular responses were determined twoweeks after the last administration by re-stimulation of splenocyteswith specific peptides as described elsewhere and detection of IFNγrelease from the splenocytes by ELISPOT assay.

ELISPOT Assay Method.

The Mouse IFN-Gamma-Kit (BD Biosciences, Catalog No. 551083) was usedfor the ELISPOT assay. The assay was performed according to themanufacturer's instructions. Briefly, plates were coated with thecapture antibody the day prior to splenocyte isolation. After isolation,cells were transferred to the ELISPOT plates and stimulated withdifferent peptides (see Table 3) for 20 hours at 37° C. IFNγ productionwas detected using the detection antibody. Plates were developed usingthe BD™ ELISPOT AEC Substrate Set (BD Biosciences, Catalog No. 551951)according to the manufacturer's instructions.

ELISPOT Stimulation Plan.

All conditions were tested in duplicate. RSV-2 and RSV-5 peptides (seeTable 8) were used at a final concentration of 5 μg/ml (1 μg/well) tostimulate 5×10⁵ and 2.5×10⁵ splenocytes per well. MVA (immunizationcontrol) was used at a Multiplicity of Infection (MOI) of 10 tostimulate 5×10⁵ and 2.5×10⁵ splenocytes per well and Concanavalin A(ConA [positive control]) was used at a final concentration of 0.5 μg/mlto stimulate 2.5×10⁵ splenocytes. As a negative control, 5×10⁵splenocytes were cultured in medium only (RPMI-1640 supplemented withGlutamax, penicillin, streptomycin, 10% Fetal Calf Serum and 10⁵Mβ-mercaptoethanol.

TABLE 8 RSV-Specific Stimulation Peptide Name SpecificityPeptide Sequence RSV-2 F KYKNAVTEL (SEQ ID NO: 20) RSV-5 M2SYIGSINNI (SEQ ID NO: 27)Analysis of BAL Fluid:

Two slides were prepared by cytospin centrifugation (800 rpm, 5 minutes)of 100 μl of BAL fluid. Slides were dried overnight and then stained.Slides were analyzed by microscopy to determine the percentage ofeosinophils and neutrophils. The rest of the BAL was then be centrifuged(12,000 rpm 5 minutes). After preparation, the BAL supernatants werestored at −20° C. (±5° C.) until analysis. IL-4 and IL-5 levels weremeasured in bronchoalveolar lavage (BAL) supernatant using commerciallyavailable ELISA kits (mIL4 PLATINUM ELISA from eBIOSCIENCE Cat N^(o)BMS613 and READY-SET-GO MIL-5 ELISA from eBIOSCIENCE Cat N^(o)88-7054-22).

Analysis of RSV Load in the Lung

The RSV load in the lung samples was determined by RSV plaque assay andby RT-qPCR.

RSV Plaque Assay.

One half each of the snap-frozen lungs was homogenized in 1 ml coldmedium using a French Press (Dulbecco's Modified Eagle Mediumsupplemented with 7% Fetal Calf Serum). After a brief centrifugation,two tubes of each supernatant were titrated in two-fold serial dilutionsonto Vero cell monolayers grown in 48-well flat-bottomed plates. Sixdays later, the monolayers were washed and fixed with 1% Formaldehyde.After 24 hours, the monolayers were stained with 0.04% Neutral Red andplaques were counted.

RSV RT-qPCR.

100 μl of the homogenized lung tissue was removed immediately and RNAwas isolated using the RNeasy® Mini Kit from Qiagen (Catalog No. 74104).The reverse transcription reaction was performed using the High CapacityRNA-to-cDNA Kit from Applied Biosystems (Catalog No. 4387406). PCRspecific for the RSV L gene was performed with the following parametersin a thermal cycler: (1) 50° C. for 2 minutes; (2) 95° C. for 10minutes; (3) 45 cycles of (15 seconds at 95° C., 1 minute at 60° C.)using the Universal PCR Master Mix from Applied Biosystems (Catalog No.4352042) and a mixture of three primers: (1) primer 1 (5′-GAA CTC AGTGTA GGT AGA ATG TTT GCA-3′; SEQ ID NO:36); (2) primer 2 (5′-TTC AGC TATCAT TTT CTC TGC CAA T-3′; SEQ ID NO:37); and (3) probe 6 (5′-TTT GAA CCTGTC TGA ACA TTC CCG GTT-3′; (SEQ ID NO:38). Copy number was determinedfrom a standard curve of pMISC202 plasmid vector containing a fragmentof the RSV L gene. Similar reactions for murine beta-actin were used asinternal controls for input cDNA using a VIC/MGB-labeled probe fromApplied Biosystems (Catalog No. 4351315).

Study Documentation.

An in-life phase flow chart was prepared to collect all informationduring the individual steps of the in-life phase. In addition, mouse- orcage-specific information was recorded on the corresponding cage card.Cage cards are not considered as study raw data but a requirement fromthe Government of Upper Bavaria.

An analysis phase flow chart was prepared to collect all informationduring the individual steps of the analysis phase. Assays weredocumented in assay-specific test records or Laboratory Note Books;cross-references were documented in the analysis phase flow chart. Allassay documentation including raw data was reviewed according tostandard procedures. In addition, sample tracking sheets for serumsamples were prepared according to standard procedures.

Data Processing.

The raw data were transferred into the corresponding Excel files forfurther analysis according to standard procedures.

ELISA.

Mean values of the OD and standard errors of the mean were calculatedusing Excel.

PRNT.

Plaques were transfer to a macro to calculate a PRNT titer according tostandard procedures.

ELISPOT.

ELISPOT plates were read with a CTL reader according to themanufacturer's instructions. The number of spot forming cells (SFC) wasdetermined for each well and transferred into an Excel file for furtherevaluation. From the incubation with 5×10⁵ and 2.5×10⁵ cells per well,the number of spots per 1×10⁶ splenocytes was calculated for each well.The mean for the negative control was calculated and was subtracted fromeach individual value prior to the calculation of the mean value permouse to obtain the Stimulation Index (SI) value (peptide-specificfrequency of IFN-γ releasing splenocytes) per mouse.

For the peptide stimulations, SI was obtained from the wells with 5×10⁵and 2.5×10⁵ cells, except when the spots were too numerous to count orfor the RSV immunized animals. In those cases only the concentration2.5×10⁵ was used. For MVA-BN stimulation, SI was obtained from the wellswith 5×10⁵, except when the spots were too numerous to count. In thatcase the concentration 2.5×10⁵ was used. Following determination of theSI for individual animals, the mean of SI (SFC per 1×10⁶ splenocytes)and standard error of the mean (SEM) was calculated per group.

RSV Plague Assay.

The numbers of plaques were counted in the well with the three highestcountable dilutions of virus. The average number of plaques adjusted bythe dilution factor was then multiplied by 10 to obtain the titer of thesolution in pfu/ml and finally multiplied by 2 to obtain the titer perlung.

RSV RT-qPCR.

PCR amplifications were measured in real time using the ABI 7500 fromApplied Biosystems (Catalog No. 4351107) and analyzed using the SystemSoftware supplied by Applied Biosystems. All values were compared to theL gene standard and were normalized to the murine beta-actindetermination for each sample.

Cytokines ELISA.

Cytokine concentrations were determined from the standard curve of therespective ELISA kits.

Results

Analysis of the Humoral Immune Response:

For both RSV-specific IgG (ELISA, FIG. 13) and RSV-specific neutralizingantibody responses (PRNT, FIG. 14) we did not observe any differencesbetween the three constructs (MVA-mBN199B, MVA-mBN201B and MVA-mBN294A)

Analysis of the Cellular Immune Response:

As expected, MVA-mBN294A had a similar T-cell response pattern thanMVA-mBN201B (FIG. 15), inducing both F and M2 specific responsesdominated by the M2 T-cell response. In contrast, MVA-mBN199B onlyinduced a F-specific response but at a higher level than MVA-mBN201B andMVA-mBN294A.

Analysis of the RSV Load in the Lungs:

RSV Challenge with RSV A2 Strain.

Mice were challenged intranasally with 10⁶ pfu of RSV(A2) two weeksafter the last immunization. Four days post-challenge, mice weresacrificed. After lung lavage with 1 ml PBS, lungs were removed and theRSV load in lung was determined by plaque assay and RT-qPCR conducted asdescribed above.

RSV Load Measured by Plaque Assay.

Four days post challenge an average of 29842 pfu per lung for thenon-immunized mice was detected (FIG. 16). As in the RSV-immunizedcontrol group, no RSV A2 plaques were detected in the lungs of animalsimmunized with MVA-mBN199B, MVA-mBN201B or MVA-mBN294A after 2 s.c.applications.

RSV Load Measured by Quantitative Real-Time PCR.

The RSV load in lung was also analyzed by RT-qPCR (FIG. 17). While RSVwas not detected by plaque assay in any of the vaccinated mice, RSVgenomes were still detectable in mice immunized s.c. twice withMVA-mBN199B, MVA-mBN201B or MVA-mBN294A. For MVA-mBN199B, the RSV loadwas 45 times lower compared to the TBS control group. RSV genomes werealso detectable for MVA-mBN201B and MVA-mBN294A but the load wasstrongly reduced compared to MVA-mBN199B, 416 times and 281 times lowercompared to the TBS control group, respectively.

Analysis of the Enhanced Disease Signs

In contrast to the batch of FI-RSV used in the experiments described inExample 3, the new batch used in this study did not show any increase ofIL-4 or IL-5 production. However we were able with this batch to detecteosinophil and neutrophil infiltrations in the BAL fluid which is themain hallmark of enhanced diseases for FI-RSV. No signs of enhanceddiseases were detectable for MVA-mBN199B, MVA-mBN201B, and MVA-mBN294A

DISCUSSION AND CONCLUSIONS

Despite the differences between MVA-mBN294A (equivalent to MVA-mBN294B)and MVA-mBN201B, both induced similar B- and T-cell responses and offersimilar protection without inducing enhanced disease. Both constructsinduced a better protection than MVA-mBN199B which expressed onlyantigenic determinants of membrane glycoproteins (F and G).

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

The invention claimed is:
 1. A recombinant modified vaccinia virusAnkara (MVA) comprising at least one nucleotide sequence encoding anantigenic determinant of a respiratory syncytial virus (RSV) membraneglycoprotein F, wherein the nucleotide sequence encodes for the aminoacid sequence of SEQ ID NO: 6; and wherein the nucleotide sequence hasat least about a 95% identity with the nucleotide sequence of SEQ ID NO:5.
 2. The recombinant MVA of claim 1, wherein the at least onenucleotide sequence encoding an antigenic determinant of the respiratorysyncytial virus (RSV) membrane glycoprotein F comprises the nucleotidesequence of SEQ ID NO:
 5. 3. The recombinant MVA of claim 1 furthercomprising at least one nucleotide sequence encoding an antigenicdeterminant of an RSV M2 matrix protein.
 4. The recombinant MVA of claim1, wherein the recombinant MVA further comprises a nucleotide sequenceencoding an antigenic determinant of an RSV N nucleocapsid protein. 5.The recombinant MVA of claim 1 further comprising at least onenucleotide sequence encoding an antigenic determinant of an RSV Gmembrane glycoprotein.
 6. The recombinant MVA of claim 5, wherein thenucleotide sequence encoding an antigenic determinant of the RSV Gmembrane glycoprotein is from an RSV strain A, strain A2, or strain B.7. The recombinant MVA of claim 5, wherein the nucleotide sequenceencoding an antigenic determinant of the RSV G membrane glycoprotein isfrom an RSV strain A2 or B.
 8. The recombinant MVA of claim 5, whereinthe nucleotide sequence encoding an antigenic determinant of the RSV Gmembrane glycoprotein comprises a nucleotide sequence encoding the aminoacid sequence selected from SEQ ID NO:2 and SEQ ID NO:8.
 9. Therecombinant MVA of claim 5, wherein the nucleotide sequence encoding anantigenic determinant of the RSV G membrane glycoprotein comprises thenucleotide sequence selected from SEQ ID NO:1 and SEQ ID NO:7.
 10. Therecombinant MVA of claim 5, further comprising an antigenic determinantof a second RSV G membrane glycoprotein.
 11. A recombinant modifiedvaccinia virus Ankara (MVA) comprising at least one nucleotide sequenceencoding an antigenic determinant of a respiratory syncytial virus (RSV)membrane glycoprotein F comprising the nucleotide sequence of SEQ IDNO:5 or a variant thereof with at least about 95% identity with thenucleotide sequence of SEQ ID NO:5 and at least one nucleotide sequenceencoding an antigenic determinant of an RSV M2 matrix protein.
 12. Therecombinant MVA of claim 11, wherein the at least one nucleotidesequence encoding an antigenic determinant of the respiratory syncytialvirus (RSV) membrane glycoprotein F comprises the nucleotide sequence ofSEQ ID NO:5.
 13. A recombinant modified vaccinia virus Ankara (MVA)comprising: a) a nucleotide sequence encoding an antigenic determinantof at least one respiratory syncytial virus (RSV) membrane glycoproteinF; b) at least one RSV membrane glycoprotein G; c) a nucleotide sequenceencoding an antigenic determinant of the RSV nucleocapsid protein N; and(c) d) a nucleotide sequence encoding an antigenic determinant of theRSV M2 matrix protein; wherein both the antigenic determinant of the RSVN nucleocapsid and of the RSV M2 matrix protein are encoded by a singleopen reading frame separated by a self-cleavage protease domain; andwherein the single open reading frame separated by a self-cleavageprotease domain comprises a nucleotide sequence encoding the amino acidsequence of SEQ ID NO:18.
 14. The recombinant MVA of claim 13, whereinthe single open reading frame separated by a self-cleavage proteasedomain comprises the nucleotide sequence of SEQ ID NO:17.
 15. Therecombinant MVA of claim 13, wherein the nucleotide sequence encoding anantigenic determinant of at least one RSV membrane glycoprotein Fencodes for the amino acid sequence of SEQ ID NO:6.
 16. The recombinantMVA of claim 15, wherein the nucleotide sequence encoding an antigenicdeterminant of at least one RSV membrane glycoprotein G encodes for theamino acid sequence of SEQ ID NO:2.
 17. The recombinant MVA of claim 16,further comprising an additional nucleotide sequence encoding anantigenic determinant of at least one RSV membrane glycoprotein G,wherein the nucleotide sequence encodes for the amino acid sequence ofSEQ ID NO:8.